Systems, methods and compositions for optical stimulation of target cells

ABSTRACT

Methods, systems and devices are implemented in connection with light-responsive ion channel molecules. One such method is implemented using a light- activated ion channel molecule that responds to a light stimulus. The method includes engineering the light-activated ion channel molecule in a cell; and activating the ion channel molecule, in response to light stimulus that is provided to the ion channel molecule and that has properties that do not activate a ChR2 ion channel, to allow ions to pass through the light-activated ion channel molecule.

RELATED DOCUMENTS

This patent document claims the benefit, under 35 U.S.C. §119(e), ofU.S. Provisional Patent Application Ser. No. 61/047,219 filed on Apr.23, 2008 and entitled “Systems, Methods and Compositions for OpticalStimulation of Target Cells;” the underlying provisional application isfully incorporated herein by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewith,and identified as follows: One 8,345 Byte ASCII (Text) file named“STFD_(—)212PCT_ST25.txt” created on Apr. 7, 2009.

OVERVIEW

The stimulation of various cells of the body has been used to produce anumber of beneficial effects. One method of stimulation involves the useof electrodes to introduce an externally generated signal into cells.One problem faced by electrode-based brain stimulation techniques is thedistributed nature of neurons responsible for a given mental process.Conversely, different types of neurons reside close to one another suchthat only certain cells in a given region of the brain are activatedwhile performing a specific task. Alternatively stated, not only doheterogeneous nerve tracts move in parallel through tight spatialconfines, but the cell bodies themselves may exist in mixed, sparselyembedded configurations. This distributed manner of processing seems todefy the best attempts to understand canonical order within the CNS, andmakes neuromodulation a difficult therapeutic endeavor. Thisarchitecture of the brain poses a problem for electrode-basedstimulation because electrodes are relatively indiscriminate withregards to the underlying physiology of the neurons that they stimulate.Instead, physical proximity of the electrode poles to the neuron isoften the single largest determining factor as to which neurons will bestimulated. Accordingly, it is generally not feasible to absolutelyrestrict stimulation to a single class of neurons using electrodes.

Another issue with the use of electrodes for stimulation is that becauseelectrode placement dictates which neurons will be stimulated,mechanical stability is frequently inadequate, and results in leadmigration of the electrodes from the targeted area. Moreover, after aperiod of time within the body, electrode leads frequently becomeencapsulated with glial cells, raising the effective electricalresistance of the electrodes, and hence the electrical power deliveryrequired to reach targeted cells. Compensatory increases in voltage,frequency or pulse width, however, may spread the electrical current andincrease the unintended stimulation of additional cells.

Another method of stimulus uses photosensitive bio-molecular structuresto stimulate target cells in response to light. For instance, lightactivated proteins or molecules can be used to control the flow of ionsthrough cell membranes. By facilitating or inhibiting the flow ofpositive or negative ions through cell membranes, the cell can bebriefly depolarized, depolarized and maintained in that state, orhyperpolarized. Neurons are an example of a type of cell that uses theelectrical currents created by depolarization to generate communicationsignals (i.e., nerve impulses). Other electrically excitable cellsinclude skeletal muscle, cardiac muscle, and endocrine cells. Neuronsuse rapid depolarization to transmit signals throughout the body and forvarious purposes, such as motor control (e.g., muscle contractions),sensory responses (e.g., touch, hearing, and other senses) andcomputational functions (e.g., brain functions). Thus, the control ofthe depolarization of cells can be beneficial for a number of differentpurposes, including (but not limited to) psychological therapy, musclecontrol and sensory functions.

Aspects of the invention are directed to photosensitive bio-molecularstructures and related methods. The present invention is exemplified ina number of implementations and applications, some of which aresummarized below.

According to one example embodiment of the present invention, animplantable arrangement is implemented having a light-generation devicefor generating light. The arrangement also has a biological portion thatmodifies target cells for stimulation in response to light generated bythe light-generation means in vivo.

According to another example embodiment of the present invention, targetcells are stimulated using an implantable arrangement. The arrangementincludes an electrical light-generation means for generating light and abiological portion. The biological portion has a photosensitivebio-molecular arrangement that responds to the generated light bystimulating target cells in vivo. Stimulation may be manifested aseither up-regulation, or down-regulation of activity at the target.

According to another example embodiment of the present invention, animplantable device delivers gene transfer vector, such as a virus, whichinduces expression of photosensitive bio-molecular membrane proteins.The device has a light generator, responsive to (for example, charged byor triggered by) an external signal, to generate light and a biologicalarrangement that includes the photosensitive bio-molecular protein thatresponds to the generated light by interacting with target cells invivo. In this manner, the electronic portions of the device may be usedto optically stimulate target cells. Stimulation may be manifested aseither up-regulation (e.g., increased neuronal firing activity), ordown-regulation (e.g., neuronal hyperpolarization, or alternatively,chronic depolarization) of activity at the target.

According to another example embodiment of the present invention, amethod is implemented for stimulating target cells using photosensitiveproteins that bind with the target cells. The method includes a step ofimplanting the photosensitive proteins and a light generating devicenear the target cells. The light generating device is activated and thephotosensitive protein stimulates the target cells in response to thegenerated light.

Applications include those associated with any population ofelectrically-excitable cells, including neurons, skeletal, cardiac,smooth muscle cells, and insulin-secreting pancreatic beta cells. Majordiseases with altered excitation-effector coupling include heartfailure, muscular dystrophies, diabetes, pain, cerebral palsy,paralysis, depression, and schizophrenia. Accordingly, the presentinvention has utility in the treatment of a wide spectrum of medicalconditions, from Parkinson's disease and brain injuries to cardiacdysrhythmias, to diabetes, and muscle spasm.

According to other example embodiments of the present invention, methodsfor generating an excitation neuron-current flow involve, in a neuron,engineering a protein that responds to light by producing an excitationcurrent to encourage depolarization of the neuron. In one such method,the protein is derived from Volvox carteri.

The above summary of the present invention is not intended to describeeach illustrated embodiment or every implementation of the presentinvention. The figures and detailed description that follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings, in which:

FIG. 1A shows a pheroidal alga Volvox carteri, consistent with anexample embodiment of the present invention;

FIG. 1B shows all-trans retinal Schiff base and related proteinsequences, consistent with an example embodiment of the presentinvention;

FIG. 1C evoked photocurrents relative to light intensity, consistentwith an example embodiment of the present invention;

FIG. 1D shows an inwardly rectifying current-voltage relationship,consistent with an example embodiment of the present invention;

FIG. 1E shows membrane currents relative to specific ions, consistentwith an example embodiment of the present invention;

FIG. 1F shows activation percentage relative to optical wavelength,consistent with an example embodiment of the present invention;

FIG. 2A shows neurons expressing VChR1-EYFP and exhibitingmembrane-localized EYFP fluorescence, consistent with an exampleembodiment of the present invention;

FIG. 2B shows VChR1-EYFP neurons photocurrents when illuminated with 531nm and 589 nm light, consistent with an example embodiment of thepresent invention;

FIG. 2C shows whole-cell inward currents for 531 nm and 589 nm light,consistent with an example embodiment of the present invention;

FIG. 2D shows twenty 5 ms light pulses delivered to VChR1-EYFP neuronsin current clamp at various frequencies, consistent with an exampleembodiment of the present invention;

FIG. 2E shows the percentages of successful spikes at variousfrequencies, consistent with an example embodiment of the presentinvention;

FIG. 2F shows that increasing frequencies of light pulses deliveredincreased steady-state depolarization, consistent with an exampleembodiment of the present invention;

FIG. 2G shows the membrane resistance, consistent with an exampleembodiment of the present invention;

FIG. 2H shows resting membrane potential, consistent with an exampleembodiment of the present invention;

FIG. 3A shows voltage responses to optical stimulation at differentwavelengths, consistent with an example embodiment of the presentinvention;

FIG. 3B shows a percentage of successful spikes for optical stimulationat different wavelengths and intensities, consistent with an exampleembodiment of the present invention;

FIG. 3C shows a percentage of successful spikes for optical stimulationat different wavelengths and intensities, consistent with an exampleembodiment of the present invention;

FIG. 4A-4D shows direct optical inhibition of local subthalamic nucleus(STN) neurons;

FIG. 5A-5C shows targeting astroglia within the STN;

FIG. 6A-6C shows optical depolarization of STN neurons at differentfrequencies;

FIG. 7A-7C shows quantification of the tissue volume recruited byoptical intervention;

FIG. 8A-8C shows selective optical control of afferent fibers in theSTN;

FIG. 9A-9D shows selective optical stimulation of layer V neurons inanterior primary motor cortex;

FIG. 10A-10C shows substantia nigra lesion and cannula track;

FIG. 11A-11C shows an additional histological characterization;

FIG. 12A-12D shows additional behavioral results;

FIG. 13A-13D shows additional electrophysiological results;

FIG. 14A-14D shows high-temporal resolution optrode traces;

FIG. 15A-15C shows latency of M1 response to optical stimulation of STN;and

FIG. 16A-16F shows changes in frequency characteristics of neuronalactivity produced by optical stimulation.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be useful for facilitatingpractical applications of a variety of photosensitive bio-molecularstructures, and the invention has been found to be particularly suitedfor use in arrangements and methods dealing with cellular membranevoltage control and stimulation. While the present invention is notnecessarily limited to such applications, various aspects of theinvention may be appreciated through a discussion of various examplesusing this context.

Consistent with one example embodiment of the present invention, alight-responsive protein/molecule is engineered in a cell. The proteinaffects a flow of ions across the cell membrane in response to light.This change in ion flow creates a corresponding change in the electricalproperties of the cells including, for example, the voltage and currentflow across the cell membrane. In one instance, the protein functions invivo using an endogenous cofactor to modify ion flow across the cellmembrane. In another instance, the protein changes the voltage acrossthe cell membrane so as to dissuade action potential firing in the cell.In yet another instance, the protein is capable of changing theelectrical properties of the cell within several milliseconds of thelight being introduced.

Consistent with a more specific example embodiment of the presentinvention, a protein, herein identified as VChR1, from Volvox carteri isused for temporally-precise optical control of neural activity. VChR1allows for selective excitation of single action potentials includingthe generation of rapid spike trains and sustained blockade of spikingover many minutes. The action spectrum of VChR1 is strongly red-shiftedrelative to ChR2 but operates at similar light power, and functions inmammals without exogenous cofactors. In one instance, VChR1 can beco-expressed with and NpHR and/or ChR2 in the target cells. Likewise,VChR1, NpHR and ChR2 can be targeted to C. elegans muscle andcholinergic motoneurons to control locomotion bidirectionally. In thisregard, VChR1, NpHR and ChR2 form an optogenetic system for multimodal,high-speed, genetically-targeted, all-optical interrogation of livingneural circuits.

Embodiments of the present invention are directed to the VChR1 protein.Various embodiments are directed toward a plasmid that contains the DNAor nucleotide sequence that expresses the VChR1 protein. Yet otherembodiments are directed toward an expression vector for expression ofthe VChR1 protein. A non-exclusive list of expression vectors includesbacterial, viral and plant plasmids. Another embodiment of the presentinvention is directed to heterologous cells that contain the VChR1protein.

Aspects of the present invention are directed toward variations of thespecific embodiment of VChR1 disclosed in FIG. 1B. One such aspectincludes mutations of the protein. These mutations may, for example,target portions of the VChR1 protein that shift or otherwise change thewavelength of light that activates the protein.

Fast light-activated microbial proteins adapted for neuroscience,including the channelrhodopsin ChR2 and the halorhodopsin NpHR, permitmillisecond-precision optical control of genetically-defined cell typeswithin intact neural tissue. Since ChR2 is a blue light-driven cationchannel that can activate neurons, and NpHR is a yellow light-drivenchloride pump that can inhibit neurons, the combination of these twoproteins allows independent neural excitation and inhibition in the samepreparation. A third major optogenetic tool, namely a second cationchannel with action spectrum significantly shifted relative to ChR2,would allow experimental testing of the differential contribution orinteraction of two distinct cell types in circuit computation orbehavior.

One ChR2-related sequence from the spheroidal alga Volvox carteri (FIG.1A) has been described, but the absorption spectrum of the protein andthe photocycle dynamics are virtually identical to those of ChR2. Asecond Volvox ChR more related to ChR1 from Chlamydomonas reinhardtii(FIG. 1B) was discovered. This new protein and variants thereof areherein referred to as VChR1.

In an experimental test, VChR1 was expressed in Xenopus oocytes andHEK293 cells, and found to evoke photocurrents were similar to those ofChR1 from Chlamydomonas. The photocurrents were graded with lightintensity, and displayed inactivation from a fast peak toward a slightlyreduced stationary plateau (FIG. 1C). The peak appeared preferentiallyat light of relatively high intensity, likely attributable to increasedaccumulation of an expected late non-conducting photocycle intermediate(FIG. 1C), and as light intensity approached saturation the evokedcurrent displayed a characteristic minimum before steady-state isreached. VChR1 exhibited an inwardly rectifying current-voltagerelationship (FIG. 1D) and under neuronal physiological conditionsconducted chiefly Na+but also H+, K+, and Ca2+ (FIG. 1E).

Primary-structural differences between VChR1 and the Chlamydomonas ChRswere identified to allow for prediction of the altered absorptionproperties (FIG. 1B, depicting SEQ. ID. NOs. 001 (ChR2), 002 (ChR1) and003 (VChR1), with primary- structural differences highlighted). First,based on previous calculations of the electrostatic potential ofbacteriorhodopsin (BR, absorption maximum at 570 nm) and sensoryrhodopsin II (SRII, absorption maximum at 500 nm) and on additionalquantum mechanical-molecular mechanical calculations (QM/MM), thecounterion complex of the cofactor all-trans retinal Schiff base (RSB;FIG. 1B) will likely be most critical for color tuning,photoisomerization and photocycle dynamics. Based on homology with othermicrobial opsin genes for which the 3D structure is known, thecounterion complex in ChR2 should be defined by R120, E123, and D253.However, these residues are fully conserved in both ChR1 and VChR1 (FIG.1B, highlighted columns 104). Second, theoretical calculations in linewith previous mutational experiments predict that three residues of theRSB binding pocket could significantly contribute to absorptiondifferences among microbial rhodopsin proteins. These amino acids areG181, L182, and S256 in ChR2 (FIG. 1B sequence, highlighted columns106); the former two are expected to be located near the RSB β-iononering (FIG. 1B structure, 108) and may, in conjunction with C183,determine absorbance spectrum, while S256 is instead likely adjacent tothe protonated nitrogen of the RSB (FIG. 1G structure, 110). In VChR1the β-ionone ring end of the RSB is expected to be more polar than inChR1 and ChR2, since the two positions 181 and 183 have been substitutedwith a polar Ser, while conversely the RSB nitrogen environment isactually less polar with an Ala at position 256. The combination ofthese three exchanges at positions 181, 183, and 256 resulting in anexpectation of a redistribution of positive charge along the RSB polyenesystem and a substantial redshift, likely by more than 40 nm, in VChR1compared to ChR2.

Three other amino acids H114, E235, and E245 (FIG. 1B, highlightedcolumns 102) are expected to modulate the RSB charge distribution bylong-range coupling, and here the H114N exchange in both VChR1 and ChR1is further predicted to increase the RSB potential at the β-ionone end.The 495 nm absorbance maximum of ChR1 (which does not express well inneurons), is indeed slightly red-shifted from that of ChR2, but thecombination of many significant changes in VChR1 predicted a robustwavelength shift on a scale useful for defining a new class of tool forneuroscience.

To initially probe the wavelength-dependence, VChR1-expressing oocyteswere excited using 10 ns laser flashes across a range of wavelengths, toallow delineation of a markedly red-shifted action spectrum thatrevealed a maximum at ˜535 nm and a small shoulder at lower wavelengthsconsistent with a second isoform peaking at 505 nm (FIG. 1F). Alentivirus carrying the alpha-CaMKII promoter to drive strong proteinexpression was constructed to test the function of VChR1 in neurons. Tovisualize VChR1 expression, the seven transmembrane domains of VChR1(residues 1-300, based on homology with the first 315 residues of ChR2)were in-frame fused to the amino-terminus of yellow fluorescent protein(VChR1-EYFP). Neurons expressing VChR1-EYFP exhibited clearlymembrane-localized EYFP fluorescence similar to that reported previouslyfor ChR2-EYFP (FIG. 2A), with expression level slightly weaker comparedwith ChR2-EYFP using the same lentiviral alpha-CaMKII expression vector.Nevertheless, VChR1-EYFP neurons exhibited strong photocurrents whenilluminated with 531 nm and even 589 nm light (FIG. 2B). Mean whole-cellinward currents were 208.8±22.3 pA (mean ±s.e.m. reported unlessotherwise stated, n=20) and 177.6±24.7 pA (n=10) when illuminated with15 mW/mm2 of 531 nm light and 13.8 mW/mm2 of 589 nm light at the sample,respectively (FIG. 2C). Apparent time constants for the rise of thephotocurrent were faster when closer to the wavelength of maximumactivation due to the shift in absorption coefficient, withcorresponding values of τ531_on=2.8±0.3 ms and τ589_on=8.0±0.7 ms (n=11for 531 nm and n=10 for 589 nm). The corresponding decay time constantswere τ531_off=133.4±11.7 ms (n=11) and τ589_off=135.7±9.8 ms (n=10).

The frequency dependence of VChR1 in evoking spikes was explored usingtrains of twenty 5 ms light pulses at 531 nm or 589 nm delivered toVChR1-EYFP neurons in current clamp (exemplar traces from 589 nmexcitation in FIG. 2D). At up to 10 Hz, more than 90% of tested cellsfired 100% of the action potentials in the train at either wavelength,and at 20 Hz cells typically fired in response to ˜65% of light pulses(FIG. 2E). In these strongly expressing cells, reliable spiking could bedriven up to 30 Hz (FIG. 2D; pyramidal neurons in culture typicallycannot follow 50 Hz or beyond in response to either current injection orChR2 photostimulation), and at 531 nm, doublets of spikes wereoccasionally evoked for each light pulse, most likely due to the slowerτoff decay constant of 133 ms compared to 12 ms for ChR210. As withChR2, VChR1 could also trigger EPSP-like subthreshold depolarizationswith lower stimulation light intensities. Delivery of light pulsebarrages evoked typical summation of the subthreshold membrane voltagechanges, with increasing frequencies of light pulses deliveringincreased steady-state depolarization (FIG. 2F).

To test for possible effects on membrane integrity, the membraneresistance and resting membrane potential were compared (FIGS. 2G and2H) by whole-cell patch clamp, among 1) VChR1-EYFPexpressing, 2)non-transduced, and 3) VChR1-EYFP-expressing neurons first patch-clamped24 hr after exposure to a typical light pulse protocol (1 s of 20 Hz, 5ms light flashes, once per minute, for 10 minutes). All cells recordedexhibited comparable values, suggesting that VChR1-EYFP expression didnot significantly alter membrane electrical properties. Subcellulardistribution appeared similar to that of ChR2, with strong membranelocalization, and VChR1 was well tolerated by these neurons. Moreover,as with ChR2 and NpHR, no all-trans retinal supplementation was neededwith after VChR1 transduction in neurons; these genes all encodemicrobial opsins, which require incorporation of all-trans retinal toform the RSB and become functional rhodopsins, but vertebrate neuronsconsistently have been found to convert expressed opsins into functionalproteins without supplementation of chemical cofactors.

Testing was performed as to whether the pronounced spectral separationbetween ChR2 and VChR1 activation would be sufficient to enableseparable activation using two different wavelengths of light. Basedupon the action spectra (FIG. 1F), 406 nm and 589 nm were selected aslikely optimal excitation wavelengths to probe separable activation ofChR2 and VChR1. For neurons expressing either ChR2 or VChR1, testing wasperformed for evoked action potentials in response to trains of twenty 5ms light pulses (406 nm and 589 nm) delivered at 5 Hz. Each wavelengthwas tested at several different light intensities to determineparameters that maximize ChR2 activation while minimizing VChR1activation at 406 nm, and vice versa. It was discovered that ChR2 andVChR1 neurons can be separately activated by 406 nm and 589 nm lightrespectively (FIG. 3A). In fact, no ChR2 neurons fired action potentialswhen illuminated with 589 nm light pulses since the absorption ispractically zero at this wavelength, whereas VChR1 neurons firedreliably at this wavelength. Conversely all ChR2 neurons fired 20 actionpotentials when illuminated with 406 nm light, at all three lightintensities (n=10, FIG. 3B). While VChR1 cells were capable of firingoccasional action potentials in response to 406 nm flashes (generally,all rhodopsins exhibit some absorption at this wavelength due totransition to the second electronic state, S0→S2 transition), thepercentage of spikes could be reduced to 13±9% when the 406 nm lightintensity was reduced to 1.2 mW/mm2 (n=10, FIG. 3C), an intensity whichcontinued to reliably and robustly drive spiking in the ChR2 neurons.

As currently implemented, the simultaneous application of VChR1 and ChR2could be used to test progressive recruitment of different cellpopulations in controlling circuit behavior. For example, two differentinterneuron or neuromodulatory populations could be recruited instepwise fashion: first by isolating population A with yellow light,followed by driving the combination of populations A and B with addedblue light. This kind of experiment has been the primary driving forcebehind developing wavelength-shifted channelrhodopsins, as thecomplexity of neural information processing and interactions ofdifferent neuromodulatory systems will require fast optical excitationat more than one wavelength to test the importance of combinatorialcomputations and modulatory gating in neural circuit dynamics andbehavior.

While the role of single cell types can be tested in separateexperiments, for convenience in some experiments it might be useful todrive two isolated populations at different times. For this kind ofexperiment, an optimal strategy would entail use of the minimum 406 nmand 589 nm light intensities sufficient to separately trigger reliableChR2 and VChR1 spikes respectively, which will simply requireindependent calibration in each experimental preparation (as in FIGS.3A-C). A cross-taper of light colors can also be employed using amonochrometer or multiple filters; at yellow wavelengths, theVChR1-labeled population will be exclusively controlled, and as theexcitation wavelength becomes progressively more blue beyond 535 nm, thecontribution of the ChR2-labeled population will become steadily moredominant (FIG. 1F). Molecular refinements (e.g., blueshifting ChR2 andnarrowing the spectrum of VChR1) can be implemented to provide furtherseparation at the blue end of the spectrum.

The identification and characterization of VChR1 for yellow-light neuralexcitation here defines the third major functionally distinct categoryof fast optogenetic tools available for interrogating the organizationand function of neural circuits, following the introduction of ChR2 forblue-light neural excitation and NpHR for yellow-light neuralinhibition. In addition to its functionally significant red-shiftedaction spectrum, VChR1 displays additional properties that are ofinterest, including reduced ratio of peak to steady-state current (FIGS.1C, 2B) compared with ChR2; while typically peak current magnitude inchannelrhodopsins depends on light intensity, external pH, and membranevoltage, the steady-state to peak ratio is larger for VChR1 than ChR2under all conditions we have explored.

The existence of two independently controllable excitation proteinsopens the door for a variety of applications including, but not limitedto, applications for treatment of a variety of disorders and the use ofa plurality of light-responsive proteins that can be selected so as torespond to a plurality of respective optical wavelengths. The family ofsingle-component proteins has been shown to respond to multiplewavelengths and intensities of light. Aspects of the invention allow forfurther mutations and/or searches for sequences that allow foradditional optical wavelengths and/or individually controllable proteinchannels. Variations on the optical stimulus (e.g., a wavelength,intensity or duration profile) can also be used. For instance,stimulation profiles may exploit overlaps in the excitation wavelengthsof two different ion channel proteins to allow excitation of bothproteins at the same time. In one such instance, the proteins may havedifferent levels of responsibility. Thus, in a neural application, oneset of ion channels may produce spiking at a different successpercentage relative to a second set of ion channels.

Many human applications of the present invention require governmentalapproval prior to their use. For instance, human use of gene therapy mayrequire such approval. However, similar gene therapies in neurons(non-proliferative cells that are non-susceptible to neoplasms) areproceeding rapidly, with active, FDA-approved clinical trials alreadyunderway involving viral gene delivery to human brains. This is likelyto facilitate the use of various embodiments of the present inventionfor a large variety of applications. The following is a non-exhaustivelist of a few examples of such applications and embodiments.

Addiction is associated with a variety of brain functions, includingreward and expectation. Additionally, the driving cause of addiction mayvary between individuals. According to one embodiment, addiction, forexample nicotine addiction, may be treated with optogeneticstabilization of small areas on the insula. Optionally, functional brainimaging, for example cued-state PET or fMRI, may be used to locate ahyper metabolic focus in order to determine a precise target spot forthe intervention on the insula surface.

Optogenetic excitation of the nucleus accumbens and septum may providereward and pleasure to a patient without need for resorting to use ofsubstances, and hence may hold a key to addiction treatment. Conversely,optogenetic stabilization of the nucleus accumbens and septum may beused to decrease drug craving in the context of addiction. In analternative embodiment, optogenetic stabilization of hyper metabolicactivity observed at the genu of the anterior cingulate (BA32) can beused to decrease drug craving. Optogenetic stabilization of cells withinthe arcuate nucleus of the medial hypothalamus which contain peptideproducts of pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) can also be used todecrease drug addiction behavior. For further information in thisregard, reference may be made to: Naqvi N H, Rudrauf D, Damasio H,Bechara A. “Damage to the insula disrupts addiction to cigarettesmoking.” Science. 2007 Jan. 26; 315(5811):531-534, which is fullyincorporated herein by reference.

Optogenetic stimulation of neuroendocrine neurons of the hypothalamicperiventricular nucleus that secrete somatostatin can be used to inhibitsecretion of growth hormone from the anterior pituitary, for example inacromegaly. Optogenetic stabilization of neuroendocrine neurons thatsecrete somatostatin or growth hormone can be used to increase growthand physical development. Among the changes that accompany “normal”aging, is a sharp decline in serum growth hormone levels after the4^(th) and 5^(th) decades. Consequently, physical deteriorationassociated with aging may be lessened through optogenetic stabilizationof the periventricular nucleus.

Optogenetic stabilization of the ventromedial nucleus of thehypothalamus, particularly the pro-opiomelanocortin (POMC) andcocaine-and-amphetamine-regulating transcript (CART) of the arcuatenucleus, can be used to increase appetite, and thereby treat anorexianervosa. Alternatively, optogenetic stimulation of the lateral nuclei ofthe hypothalamus can be used to increase appetite and eating behaviors.

Optogenetic excitation in the cholinergic cells of affected areasincluding the temporal lobe, the NBM (Nucleus basalis of Meynert) andthe posterior cingulate gyms (BA 31) provides stimulation, and henceneurotrophic drive to deteriorating areas. Because the affected areasare widespread within the brain, an analogous treatment with implantedelectrodes may be less feasible than an opto-genetic approach.

Anxiety disorders are typically associated with increased activity inthe left temporal and frontal cortex and amygdala, which trends towardnormal as anxiety resolves. Accordingly, the affected left temporal andfrontal regions and amygdala may be treated with optogeneticstabilization, so as to dampen activity in these regions.

In normal physiology, photosensitive neural cells of the retina, whichdepolarize in response to the light that they receive, create a visualmap of the received light pattern. Optogenetic ion channels can be usedto mimic this process in many parts of the body, and the eyes are noexception. In the case of visual impairment or blindness due to damagedretina, a functionally new retina can be grown, which uses naturalambient light rather than flashing light patterns from an implanteddevice. The artificial retina grown may be placed in the location of theoriginal retina (where it can take advantage of the optic nerve servingas a conduit back to the visual cortex). Alternatively, the artificialretina may be placed in another location, such as the forehead, providedthat a conduit for the depolarization signals are transmitted tocortical tissue capable of deciphering the encoded information from theoptogenetic sensor matrix. Cortical blindness could also be treated bysimulating visual pathways downstream of the visual cortex. Thestimulation would be based on visual data produced up stream of thevisual cortex or by an artificial light sensor.

Treatment of tachycardia may be accomplished with optogeneticstimulation to parasympathetic nervous system fibers including CN X orVagus Nerve. This causes a decrease in the SA node rate, therebydecreasing the heart rate and force of contraction. Similarly,optogenetic stabilization of sympathetic nervous system fibers withinspinal nerves T1 through T4, serves to slow the heart. For the treatmentof pathological bradycardia, optogenetic stabilization of the Vagusnerve, or optogenetic stimulation of sympathetic fibers in T1 through T4will serve to increase heart rate. Cardiac disrhythmias resulting fromaberrant electrical foci that outpace the sinoatrial node may besuppressed by treating the aberrant electrical focus with moderateoptogenetic stabilization. This decreases the intrinsic rate of firingwithin the treated tissue, and permits the sinoatrial node to regain itsrole in pacing the heart's electrical system. In a similar way, any typeof cardiac arrhythmia could be treated. Degeneration of cardiac tissuethat occurs in cardiomyopathy or congestive heart failure could also betreated using this invention; the remaining tissue could be excitedusing various embodiments of the invention.

Optogenetic excitation stimulation of brain regions including thefrontal lobe, parietal lobes and hippocampi, may increase processingspeed, improve memory, and stimulate growth and interconnection ofneurons, including spurring development of neural progenitor cells. Asan example, one such application of the present invention is directed tooptogenetic excitation stimulation of targeted neurons in the thalamusfor the purpose of bringing a patient out of a near-vegetative(barely-conscious) state. Growth of light-gated ion channels or pumps inthe membrane of targeted thalamus neurons is effected. These modifiedneurons are then stimulated (e.g., via optics which may also gain accessby the same passageway) by directing a flash of light thereupon so as tomodulate the function of the targeted neurons and/or surrounding cells.For further information regarding appropriate modulation techniques (viaelectrode-based treatment) or further information regarding theassociated brain regions for such patients, reference may be made to:Schiff N D, Giacino J T, Kalmar K, Victor J D, Baker K, Gerber M, FritzB, Eisenberg B, O'Connor J O, Kobylarz E J, Farris S, Machado A, McCaggC, Plum F, Fins J J, Rezai A R “Behavioral improvements with thalamicstimulation after severe traumatic brain injury,” Nature, Vol. 448, Aug.2, 2007, pp. 600-604.

In an alternative embodiment, optogenetic excitation may be used totreat weakened cardiac muscle in conditions such as congestive heartfailure. Electrical assistance to failing heart muscle of CHF isgenerally not practical, due to the thin-stretched, fragile state of thecardiac wall, and the difficulty in providing an evenly distributedelectrical coupling between an electrodes and muscle. For this reason,preferred methods to date for increasing cardiac contractility haveinvolved either pharmacological methods such as Beta agonists, andmechanical approaches such as ventricular assist devices. In thisembodiment of the present invention, optogenetic excitation is deliveredto weakened heart muscle via light emitting elements on the innersurface of a jacket surround the heart or otherwise against the affectedheart wall. Light may be diffused by means well known in the art, tosmoothly cover large areas of muscle, prompting contraction with eachlight pulse.

Optogenetic stabilization in the subgenual portion of the cingulate gyms(Cg25), yellow light may be applied with an implanted device. The goalwould be to treat depression by suppressing target activity in manneranalogous to what is taught by Mayberg HS et al., “Deep BrainStimulation for Treatment-Resistant Depression,” Neuron, Vol. 45,651-660, Mar. 3, 2005, pp. 651-660, which is fully incorporated hereinby reference. In an alternative embodiment, an optogenetic excitationstimulation method is to increase activity in that region in a manneranalogous to what is taught by Schlaepfer et al., “Deep Brainstimulation to Reward Circuitry Alleviates Anhedonia in Refractory MajorDepression,” Neuropsychopharmacology 2007, pp. 1-10, which is fullyincorporated herein by reference.

In yet another embodiment, the left dorsolateral prefrontal cortex(LDPFC) is targeted with an optogenetic excitation stimulation method.Pacing the LDLPFC at 5-20 Hz serves to increase the basal metaboliclevel of this structure which, via connecting circuitry, serves todecrease activity in Cg 25, improving depression in the process.Suppression of the right dorsolateral prefrontal cortex (RDLPFC) is alsoan effective depression treatment strategy. This may be accomplished byoptogenetic stabilization on the RDLPFC, or suppression may also beaccomplished by using optogenetic excitation stimulation, and pulsing ata slow rate (e.g., 1 Hz or less) improving depression in the process.Vagus nerve stimulation (VNS) may be improved using an optogeneticapproach. Use of optogenetic excitation may be used in order tostimulate only the vagus afferents to the brain, such as the nodoseganglion and the jugular ganglion. Efferents from the brain would notreceive stimulation by this approach, thus eliminating some of theside-effects of VNS including discomfort in the throat, a cough,difficulty swallowing and a hoarse voice. In an alternative embodiment,the hippocampus may be optogenetically excited, leading to increaseddendritic and axonal sprouting, and overall growth of the hippocampus.Other brain regions implicated in depression that could be treated usingthis invention include the amygdala, accumbens, orbitofrontal andorbitomedial cortex, hippocampus, olfactory cortex, and dopaminergic,serotonergic, and noradrenergic projections. Optogenetic approachescould be used to control spread of activity through structures like thehippocampus to control depressive symptoms.

So long as there are viable alpha and beta cell populations in thepancreatic islets of Langerhans, the islets can be targeted for thetreatment of diabetes. For example, when serum glucose is high (asdetermined manually or by closed loop glucose detection system),optogenetic excitation may be used to cause insulin release from thebeta cells of the islets of Langerhans in the pancreas, whileoptogenetic stabilization is used to prevent glucagon release from thealpha cells of the islets of Langerhans in the pancreas. Conversely,when blood sugars are too low (as determined manually or by closed loopglucose detection system), optogenetic stabilization may be used to stopbeta cell secretion of insulin, and optogenetic excitation may be usedto increase alpha-cell secretion of glucagon.

For treatment of epilepsy, quenching or blocking epileptogenic activityis amenable to optogenetic approaches. Most epilepsy patients have astereotyped pattern of activity spread resulting from an epileptogenicfocus. Optogenetic stabilization could be used to suppress the abnormalactivity before it spreads or truncated it early in its course.Alternatively, activation of excitatory tissue via optogeneticexcitation stimulation could be delivered in a series of deliberatelyasynchronous patterns to disrupt the emerging seizure activity. Anotheralternative involves the activation of optogenetic excitationstimulation in GABAergic neurons to provide a similar result. Thalamicrelays may be targeted with optogenetic stabilization triggered when anabnormal EEG pattern is detected.

Another embodiment involves the treatment of gastrointestinal disorders.The digestive system has its own, semi-autonomous nervous systemcontaining sensory neurons, motor neurons and interneurons. Theseneurons control movement of the GI tract, as well as trigger specificcells in the gut to release acid, digestive enzymes, and hormonesincluding gastrin, cholecystokinin and secretin. Syndromes that includeinadequate secretion of any of these cellular products may be treatedwith optogenetic stimulation of the producing cell types, or neuronsthat prompt their activity. Conversely, optogenetic stabilization may beused to treat syndromes in which excessive endocrine and exocrineproducts are being created. Disorders of lowered intestinal motility,ranging from constipation (particularly in patients with spinal cordinjury) to megacolan may be treated with optogenetic excitation of motorneurons in the intestines. Disorders of intestinal hypermotility,including some forms of irritable bowel syndrome may be treated withoptogenetic stabilization of neurons that control motility. Neurogenticgastric outlet obstructions may be treated with optogeneticstabilization of neurons and musculature in the pyloris. An alternativeapproach to hypomobility syndromes would be to provide optogeneticexcitation to stretch-sensitive neurons in the walls of the gut,increasing the signal that the gut is full and in need of emptying.

In this same paradigm, an approach to hypermobility syndromes of the gutwould be to provide optogenetic stabilization to stretch receptorneurons in the lower GI, thus providing a “false cue” that the gut wasempty, and not in need of emptying. In the case of frank fecalincontinence, gaining improved control of the internal and externalsphincters may be preferred to slowing the motility of the entire tract.During periods of time during which a patient needs to hold feces in,optogenetic excitation of the internal anal sphincter will provide forretention. Providing optogenetic stimulation to the external sphinctermay be used to provide additional continence. When the patient isrequired to defecate, the internal anal sphincter, and then externalanal sphincter should be relaxed, either by pausing the optogeneticstimulation, or by adding optogenetic stabilization.

Conductive hearing loss may be treated by the use of optical cochlearimplants. Once the cochlea has been prepared for optogeneticstimulation, a cochlear implant that flashes light may be used.Sensorineural hearing loss may be treated through optical stimulation ofdownstream targets in the auditory pathway. Another embodiment of thepresent invention is directed toward the treatment of blood pressuredisorders, such as hypertension. Baroreceptors and chemoreceptors inregions such as the aorta (aortic bodies and paraaortic bodies) and thecarotid arteries (“carotic bodies”) participate in the regulation ofblood pressure and respiration by sending afferents via the vagus nerve(CN X), and other pathways to the medulla and pons, particularly thesolitary tract and nucleus. Optogenetic excitation of the carotidbodies, aortic bodies, paraortic bodies, may be used to send a falsemessage of “hypertension” to the solitary nucleus and tract, causing itto report that blood pressure should be decreased. Optogeneticexcitation or stabilization directly to appropriate parts of thebrainstem may also be used to lower blood pressure. The oppositemodality causes the optogenetic approach to serve as a pressor, raisingblood pressure. A similar effect may also be achieved via optogeneticexcitation of the Vagus nerve, or by optogenetic stabilization ofsympathetic fibers within spinal nerves T1-T4. In an alternativeembodiment, hypertension may be treated with optogenetic stabilizationof the heart, resulting in decreased cardiac output and lowered bloodpressure. According to another embodiment, optogenetic stabilization ofaldosterone-producing cells within the adrenal cortex may be used todecrease blood pressure. In yet another alternative embodiment,hypertension may be treated by optogenetic stabilization of vascularsmooth muscle. Activating light may be passed transcutaneously to theperipheral vascular bed.

Another example embodiment is directed toward the treatment ofhypothalamic-pituitary-adrenal axis disorders. In the treatment ofhypothyroidism, optogenetic excitation of parvocellular neuroendocrine,neurons in the paraventricular and anterior hypothalamic nuclei can beused to increase secretion of thyrotropin-releasing hormone (TRH). TRH,in turn, stimulates anterior pituitary to secrete TSH. Conversely,hyperthyroidism may be treated with optogenetic stabilization of theprovocellular neuroendocrine neurons. For the treatment of adrenalinsufficiency, or of Addison's disease, optogenetic excitation ofparvocellular neuroendocrine neurons in the supraoptic nucleus andparaventricular nuclei may be used to increase the secretion ofvasopressin, which, with the help of corticotropin-releasing hormone(CRH), stimulate anterior pituitary to secrete ACTH. Cushing syndrome,frequently caused by excessive ACTH secretion, may be treated withoptogenetic stabilization of the parvocellular neuroendocrine neurons ofsupraoptic nucleus via the same physiological chain of effects describedabove. Neuroendocrine neurons of the arcuate nucleus produce dopamine,which inhibits secretion of prolactin from the anterior pituitary.Hyperprolactinemia can therefore be treated via optogenetic excitation,while hypoprolactinemia can be treated with optogenetic stabilization ofthe neuroendocrine cells of the arcuate nucleus.

In the treatment of hyperautonomic states, for example anxietydisorders, optogenetic stabilization of the adrenal medulla may be usedto reduce norepinephrine output. Similarly, optogenetic stimulation ofthe adrenal medulla may be used in persons with need for adrenalinesurges, for example those with severe asthma, or disorders that manifestas chronic sleepiness.

Optogenetic stimulation of the adrenal cortex will cause release ofchemicals including cortisol, testosterone, and aldosterone. Unlike theadrenal medualla, the adrenal cortex receives its instructions fromneuroendocrine hormones secreted from the pituitary and hypothalamus,the lungs, and the kidneys. Regardless, the adrenal cortex is amenableto optogenetic stimulation. Optogenetic stimulation of thecortisol-producing cells of the adrenal cortex may be used to treatAddison's disease. Optogenetic stabilization of cortisol-producing cellsof the adrenal cortex may be used to treat Cushing's disease.Optogenetic stimulation of testosterone-producing cells may be used totreat disorders of sexual interest in women: Optogenetic stabilizationof those same cells may be used to decrease facial hair in women.Optogenetic stabilization of aldosterone-producing cells within theadrenal cortex may be used to decrease blood pressure. Optogeneticexcitation of aldosterone-producing cells within the adrenal cortex maybe used to increase blood pressure.

Optogenetic excitation stimulation of specific affected brain regionsmay be used to increase processing speed, and stimulate growth andinterconnection of neurons, including spurring the maturation of neuralprogenitor cells. Such uses can be particularly useful for treatment ofmental retardation.

According to another embodiment of the present invention, various musclediseases and injuries can be treated. Palsies related to muscle damage,peripheral nerve damage and to dystrophic diseases can be treated withoptogenetic excitation to cause contraction, and optogeneticstabilization to cause relaxation. This latter relaxation viaoptogenetic stabilization approach can also be used to prevent musclewasting, maintain tone, and permit coordinated movement as opposingmuscle groups are contracted. Likewise, frank spasticity can be treatedvia optogenetic stabilization.

In areas as diverse as peripheral nerve truncation, stroke, traumaticbrain injury and spinal cord injury, there is a need to foster thegrowth of new neurons, and assist with their integration into afunctional network with other neurons and with their target tissue.Re-growth of new neuronal tracts may be encouraged via optogeneticexcitation, which serves to signal stem cells to sprout axons anddendrites, and to integrate themselves with the network. Use of anoptogenetic technique (as opposed to electrodes) prevents receipt ofsignals by intact tissue, and serves to ensure that new target tissuegrows by virtue of a communication set up with the developing neurons,and not with an artificial signal like current emanating from anelectrode.

Obesity can be treated with optogenetic excitation to the ventromedialnucleus of the hypothalamus, particularly the pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) of thearcuate nucleus. In an alternative embodiment, obesity can be treatedvia optogenetic stabilization of the lateral nuclei of the hypothalamus.In another embodiment, optogenetic stimulation to leptin-producing cellsor to cells with leptin receptors within the hypothalamus may be used todecrease appetite and hence treat obesity.

Destructive lesions to the anterior capsule and analogous DBS to thatregion are established means of treating severe, intractableobsessive-compulsive disorder 48 (OCD48). Such approaches may beemulated using optogenetic stabilization to the anterior limb of theinternal capsule, or to regions such as BA32 and Cg24 which showmetabolic decrease as OCD remits.

Chronic pain can be treated using another embodiment of the presentinvention. Electrical stimulation methods include local peripheral nervestimulation, local cranial nerve stimulation and “sub threshold” motorcortex stimulation. Reasonable autogenic approaches include optogeneticstabilization at local painful sites. Attention to promoter selectionwould ensure that other sensory and motor fibers would be unaffected.Selective optogenetic excitation of interneurons at the primary motorcortex also may provide effective pain relief. Also, optogeneticstabilization at the sensory thalamus, (particularly medial thalamicnuclei), periventricular grey matter, and ventral raphe nuclei, may beused to produce pain relief. In an alternative embodiment, optogeneticstabilization of parvalbumin-expressing cells targeting as targetingstrategy, may be used to treat pain by decreasing Substance Pproduction. The release of endogenous opiods may be accomplished byusing optogenetic excitation to increase activity in the nucleusaccumbens. In an alternative embodiment, when POMC neurons of thearcuate nucleus of the medial hypothalamus are optogenetically excited,beta endorphin are increased, providing viable treatment approaches fordepression and for chronic pain.

Certain personality disorders, including the borderline and antisocialtypes, demonstrate focal deficits in brain disorders including“hypofrontality.” Direct or indirect optogenetic excitation of theseregions is anticipated to produce improvement of symptoms. Abnormalbursts of activity in the amygdala are also known to precipitate sudden,unprompted flights into rage: a symptom of borderline personalitydisorder, as well as other conditions, which can benefit fromoptogenetic stabilization of the amygdala. Optogenetic approaches couldimprove communication and synchronization between different parts of thebrain, including amygdala, striatum, and frontal cortex, which couldhelp in reducing impulsiveness and improving insight.

The amygdalocentric model of post-traumatic-stress disorder (PTSD)proposes that it is associated with hyperarousal of the amygdala andinsufficient top-down control by the medial prefrontal cortex and thehippocampus. Accordingly, PTSD may be treated with optogeneticstabilization of the amygdale or hippocampus.

Schizophrenia is characterized by abnormalities including auditoryhallucinations. These might be treated by suppression of the auditorycortex using optogenetic stabilization. Hypofrontality associated withschizophrenia might be treated with optogenetic excitation in theaffected frontal regions. Optogenetic approaches could improvecommunication and synchronization between different parts of the brainwhich could help in reducing misattribution of self-generated stimuli asforeign.

Optogenetic stabilization of cells within the arcuate nucleus of themedial hypothalamus, which contain peptide products ofpro-opiomelanocortin (POMC) and cocaine-and-amphetamine-regulatingtranscript (CART), can be used to reduce compulsive sexual behavior.Optogenetic excitation of cells within the arcuate nucleus of the medialhypothalamus which contain peptide products of pro-opiomelanocortin(POMC) and cocaine-and-amphetamine-regulating transcript (CART) may beused to increase sexual interest in the treatment of cases of disordersof sexual desire. In the treatment of disorders of hypoactive sexualdesire testosterone production by the testes and the adrenal glands canbe increased through optogenetic excitation of the pituitary gland.Optogenetic excitation of the nucleus accumbens can be used for thetreatment of anorgasmia.

The suprachiasmatic nucleus secretes melatonin, which serves to regulatesleep/wake cycles. Optogenetic excitation to the suprachiasmic nucleuscan be used to increase melatonin production, inducing sleep, andthereby treating insomnia. Orexin (hypocretin) neurons strongly excitenumerous brain nuclei in order to promote wakefulness. Optogenteticexcitation of orexin-producing cell populations can be used to treatnarcolepsy, and chronic daytime sleepiness.

Optogenetic stimulation of the supraoptic nucleus may be used to inducesecretion of oxytocin, can be used to promote parturition duringchildbirth, and can be used to treat disorders of social attachment.

Like muscular palsies, the motor functions that have been de-afferentedby a spinal cord injury may be treated with optogenetic excitation tocause contraction, and optogenetic stabilization to cause relaxation.This latter relaxation via optogenetic stabilization approach may alsobe used to prevent muscle wasting, maintain tone, and permit coordinatedmovement as opposing muscle groups are contracted. Likewise, frankspasticity may be treated via optogenetic stabilization. Re-growth ofnew spinal neuronal tracts may be encouraged via optogenetic excitation,which serves to signal stem cells to sprout axons and dendrites, and tointegrate themselves with the network.

Stroke deficits include personality change, motor deficits, sensorydeficits, cognitive loss, and emotional instability. One strategy forthe treatment of stroke deficits is to provide optogenetic stimulationto brain and body structures that have been deafferented from excitatoryconnections. Similarly, optogenetic stabilization capabilities can beimparted on brain and body structures that have been deafferented frominhibitory connections.

Research indicates that the underlying pathobiology in Tourette'ssyndrome is a phasic dysfunction of dopamine transmission in corticaland subcortical regions, the thalamus, basal ganglia and frontal cortex.In order to provide therapy, affected areas are preferably firstidentified using techniques including functional brain imaging andmagnetoencephalography (MEG). Whether specifically identified or not,optogenetic stabilization of candidate tracts may be used to suppressmotor tics. Post-implantation empirical testing of device parametersreveals which sites of optogenetic stabilization, and which areunnecessary to continue.

In order to treat disorders of urinary or fecal incontinence optogeneticstabilization can be used to the sphincters, for example via optogeneticstabilization of the bladder detrussor smooth muscle or innervations ofthat muscle. When micturation is necessary, these optogenetic processesare turned off, or alternatively can be reversed, with optogeneticstabilization to the (external) urinary sphincter, and optogeneticexcitation of the bladder detrussor muscle or its innervations. When abladder has been deafferentated, for example, when the sacral dorsalroots are cut or destroyed by diseases of the dorsal roots such as tabesdorsalis in humans, all reflex contractions of the bladder areabolished, and the bladder becomes distended. Optogenetic excitation ofthe muscle directly can be used to restore tone to the detrussor,prevent kidney damage, and to assist with the micturition process. Asthe bladder becomes “decentralized” and hypersensitive to movement, andhence prone to incontinence, optogenetic stabilization to the bladdermuscle can be used to minimize this reactivity of the organ.

In order to selectively excite/inhibit a given population of neurons,for example those involved in the disease state of an illness, severalstrategies can be used to target the optogenetic proteins/molecules tospecific populations.

For various embodiments of the present invention, genetic targeting maybe used to express various optogenetic proteins or molecules. Suchtargeting involves the targeted expression of the optogeneticproteins/molecules via genetic control elements such as promoters (e.g.,Parvalbumin, Somatostatin, Cholecystokinin, GFAP), enhancers/silencers(e.g., Cytomaglovirus Immediate Early Enhancer), and othertranscriptional or translational regulatory elements (e.g., WoodchuckHepatitis Virus Post-transcriptional Regulatory Element). Permutationsof the promoter+enhancer+regulatory element combination can be used torestrict the expression of optogenetic probes to genetically-definedpopulations.

Various embodiments of the present invention may be implemented usingspatial/anatomical targeting. Such targeting takes advantage of the factthat projection patterns of neurons, virus or other reagents carryinggenetic information (DNA plasmids, fragments, etc), can be focallydelivered to an area where a given population of neurons project to. Thegenetic material will then be transported back to the bodies of theneurons to mediate expression of the optogenetic probes. Alternatively,if it is desired to label cells in a focal region, viruses or geneticmaterial may be focally delivered to the interested region to mediatelocalized expression.

Various gene delivery systems are useful in implementing one or moreembodiments of the present invention. One such delivery system isAdeno-Associated Virus (AAV). AAV can be used to deliver apromoter+optogenetic probe cassette to a specific region of interest.The choice of promoter will drive expression in a specific population ofneurons. For example, using the CaMKIIα promoter will drive excitatoryneuron specific expression of optogenetic probes. AAV will mediatelong-term expression of the optogenetic probe for at least one year ormore. To achieve more specificity, AAV may be pseudotyped with specificserotypes 1 to 8, with each having different trophism for different celltypes. For instance, serotype 2 and 5 is known to have goodneuron-specific trophism.

Another gene delivery mechanism is the use of a retrovirus. HIV or otherlentivirus-based retroviral vectors may be used to deliver apromoter+optogenetic probe cassette to a specific region of interest.Retroviruses may also be pseudo-typed with the

Rabies virus envelope glycoprotein to achieve retrograde transport forlabeling cells based on their axonal projection patterns. Retrovirusesintegrate into the host cell's genome, therefore are capable ofmediating permanent expression of the optogenetic probes. Non-lentivirusbased retroviral vectors can be used to selectively label dividingcells.

Gutless Adenovirus and Herpes Simplex Virus (HSV) are two DNA-basedviruses that can be used to deliver promoter+optogenetic probe cassetteinto specific regions of the brain as well. HSV and Adenovirus have muchlarger packaging capacities and therefore can accommodate much largerpromoter elements and can also be used to deliver multiple optogeneticprobes or other therapeutic genes along with optogenetic probes.

Focal Electroporation can also be used to transiently transfect neurons.DNA plasmids or fragments can be focally delivered into a specificregion of the brain. By applying mild electrical current, surroundinglocal cells will receive the DNA material and expression of theoptogenetic probes.

In another instance, lipofection can be used by mixing genetic materialwith lipid reagents and then subsequently injected into the brain tomediate transfection of the local cells.

Various embodiments involve the use of various control elements. Inaddition to genetic control elements, other control elements(particularly promoters and enhancers whose activities are sensitive tochemical, magnetic stimulation or infrared radiation) can be used tomediate temporally-controlled expression of the optogenetic probes. Forexample, a promoter whose transcriptional activity is subject toinfrared radiation allows one to use focused radiation to fine tune theexpression of optogenetic probes in a focal region at only the desiredtime.

Parkinson's Disease can be treated by expressing optogeneticstabilization in the glutamatergic neurons in either the subthalamicnucleus (STN) or the globus pallidus interna (GPi) using anexcitatory-specific promoter such as CaMKIIα, and apply optogeneticstabilization. Unlike electrical modulation in which all cell-types areaffected, only glutamatergic STN neurons would be suppressed.

Aspects of the present invention are directed towards testing a model ofa neural circuit or disease. The model can define output response of thecircuit as a function of input signals. The output response can beassessed using a number of different measurable characteristics. Forinstance, characteristics can include an electrical response ofdownstream neurons and/or behavioral response of a patient. To test themodel, optogentic probes are expressed at an input position for themodel. The optogenetic probes are stimulated and the outputcharacteristics are monitored and compared to an output predicted by themodel.

In certain implementations, the use of optogenetic probes allows forfine tuning of models defined using electrical probes. As electricalprobes provide only limited ability to direct the stimulus and thus arenot well suited for stimulus of certain areas without also directlystimulating nearby areas. Optogenetic probes disclosed herein provide amechanism for more precise selection of the stimulus location. Forinstance, the stimulus from the optogenetic probes can be directed tovery specific types of circuits/cells, such as afferent fibers. Thefollowing description provides an example implementation consistent withsuch an embodiment and is meant to show the feasibility and wide-rangingapplicability for aspects of present invention. According to oneembodiment of the present invention, the invention may be used in animalmodels of DBS, for example in Parkinsonian rats, to identify the targetcell types responsible for therapeutic effects (an area of intensedebate and immense clinical importance). This knowledge alone may leadto the development of improved pharmacological and surgical strategiesfor treating human disease.

One such application involves long-term potentiation (LTP) and/orlong-term depression (LTD) between two neural groups. By targeting theexpression of VChR1 and ChR2 to different neural populations andstimulating each with a different frequency of light, LTP or LTD can beaccomplished between the two groups. Each group can be individuallycontrolled using the respective wavelength of light. This can beparticularly useful for applications in which the spatial arrangement ofthe two groups presents issues with individual control using the samewavelength of light. Thus, the light delivery device(s) are lesssusceptible to exciting the wrong neural group and can be less reliantupon precise spatial location of the optical stimulus.

The delivery of the proteins to cells in vivo can be accomplished usinga number of different deliver devices, methods and systems. On suchdelivery device is an implantable device that delivers a nucleotidesequence for modifying cells in vivo, such as a viral-vector. Theimplantable device can also include a light delivery mechanism. Thelight delivery can be accomplished using, for example, light-emittingdiodes (LEDs), fiber optics and/or Lasers.

Another embodiment of the present invention involves the use of VChR1 inaffecting stem cell fate including survival/death, differentiation andreplication. The modulation of electrical properties has been shown tocontrol stem cell fate. Various techniques can be used to providestimulus patterns that modify stem cell fate. A specific example isconsistent with those techniques used in Deisseroth, K. et al.“Excitation-neurogenesis coupling in adult neural stem/progenitorcells,” Neuron 42, pp. 535-552 (2004), which is fully incorporatedherein by reference.

Another embodiment of the present invention is directed to the use ofVChR1 to assess the efficacy of treatments. This can include, but is notlimited to, drug screening, treatment regimens or modeling oftreatments/disorders. In a specific embodiment, VChR1 is used as theprimary optically responsive protein in such assessments. In alternateembodiments, VChR1 is used with other types of optically responsiveproteins (e.g., ChR2 and/or NpHR) that respond to different wavelengths.

A specific embodiment of the present invention involves the use of VChR1to generate a mammalian codon-optimized cDNA sequence and synthesized(DNA 2.0, Menlo Park, Calif.).

Lentiviral vector construction was accomplished using the followingmethods. VChR1-EYFP was constructed by fusing VChR1(1-300) with EYFP viaa NotI restriction site. The fusion gene was then ligated into the AgeIand EcoRI sites of alpha-CaMKII lentiviral backbone to generate thepLenti-CaMKIIα-VChR1-EYFP-WPRE vector. Construction of thepLenti-CaMKIIα-ChR2-EYFP-WPRE vector was previously described.Recombinant lentiviruses were generated. For further details regardingthe construction or use of such vectors reference can be made to Zhang,F., et al. “Multimodal fast optical interrogation of neural circuitry,”Nature 446, pp. 633-639 (2007), which is fully incorporated herein byreference.

Cultured hippocampal neurons were prepared as described in Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K.“Millisecond-timescale, genetically targeted optical control of neuralactivity,” Nat Neurosci 8, pp. 1263-1268 (2005), which is fullyincorporated herein by reference.

For whole-cell recording in cultured hippocampal neurons, theintracellular solution contained 129 mM K-Gluconate, 10 mM HEPES, 10 mMKCl, 4 mM MgATP, and 0.3 mM Na3GTP, titrated to pH 7.2. For culturedhippocampal neurons, Tyrode's solution was employed as the extracellularsolution (125 mM NaCl, 2 mM KCl, 3 mM CaCl2, 1 mM MgCl2, 30 mM glucose,and 25 mM HEPES, titrated to pH 7.3). Recordings were conducted on anupright Leica DM-LFSA microscope equipped with a 40× water-immersionobjective. Borosilicate glass (Sutter Instruments) pipette resistanceswere ˜5 MΩ, with a range of 4-6 MΩ. Access resistance was 10-30 MΩ andmonitored for stability throughout the recording. All recordings wereconducted in the presence of synaptic transmission blockers as describedin Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K.“Millisecond-timescale, genetically targeted optical control of neuralactivity,” Nat Neurosci 8, pp. 1263-1268 (2005).

For hippocampal neuron photostimulation, the following three filterswere used in the Lambda DG-4 optical switch (Sutter Instruments) with a300W Xenon lamp: 406nm (FF01-406/15-25), 531nm (FF01-531/22-25), and589nm (FF01-589/15-25) (Semrock).

For oocyte experiments, a synthetic DNA sequence corresponding toVChR11-313 (vchopl; adapted to human codon-usage, Geneart, Regensburg,Germany) was subcloned into VChR1 pGEMHE and pEGFP. cRNAs encoding ChR2and VChR1, synthesized in vitro from pGEMHEplasmid by T7 RNA polymerase(mMessage mMachine, Ambion), were injected into the oocytes (50ng/cell). The oocytes were stored for 3-7 days in the dark at 18° C. inRinger solution (96 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mMMOPS-NaOH, pH 7.5) in the presence of 1 mg/ml penicillin, 1 mg/mlstreptomycin, 1 μM all-trans-retinal, and 0.5 mM theophylline.

Two-electrode voltage clamp on Xenopus laevis oocytes were performed toobtain action spectra. 10 ns laser flashes (400-620 nm, 4-9×1019 photonss-1 m-2) from a Rainbow OPO (OPOTEK, Carlsbad, CA) pumped by the thirdharmonic of a Brilliant b Nd-YAG-Laser (Quantel, Les Ulis Cedex, France)were applied to the oocyte via a 1 mm light guide. The amplifier Tec-05X(NPI Electronic, Tamm, Germany) was compensated to keep the voltagechange below 0.05 mV at a half saturating laser flash. Data acquisitionand light triggering were controlled with pCLAMP software via DigiData1440A interfaces (Molecular Devices, Sunnyvale, USA).

The following discussion includes a detailed discussion regardingresults of an application for treatment and characterization ofParkinson's Disease (PD). This specific implementation and thecorresponding results are not meant to be limiting. To first address themost widely-held hypothesis in the field, we asked if direct,reversible, bona fide inhibition of local-circuit excitatory STN neuronswould be therapeutic in PD. The STN measures <1 mm³ in rats, buttargeting accuracy can be aided by extracellular recordings during opsinvector introduction, since STN is characterized by a particular firingpattern which is distinguishable from bordering regions (FIG. 4A, FIG.10C).

The STN is a predominantly excitatory structure embedded within aninhibitory network. This anatomical arrangement enables a targetingstrategy for selective STN inhibition (FIG. 4B), in which eNpHR isexpressed under control of the CaMKIIα promoter, selective forexcitatory glutamatergic neurons and not inhibitory cells, fibers ofpassage, glia, or neighboring structures. In this way true opticalinhibition is targeted to the dominant local neuron type within STN.

Optical circuit interventions were tested in rats that had been madehemiparkinsonian by injection of 6-hydroxydopamine (6-OHDA) unilaterallyinto the right medial forebrain bundle (MFB). Loss of nigraldopaminergic cells following 6-OHDA administration was confirmed bydecreased tyrosine hydroxylase levels unilaterally in the substantianigra pars compacta (FIG. 10A). These hemiparkinsonian rodents havespecific deficits in contralateral (left) limb use and display(rightward) rotations ipsilateral to the lesion, which increase infrequency when the subjects are given amphetamine to facilitatefunctional evaluation, and decrease in frequency upon treatment withdopamine agonists or following electrical DBS (FIG. 4D, right). Thisamphetamine-induced rotation test is widely used for identifyingtreatments in hemiparkinsonian rodents, which can be complemented withother behavioral assays such as locomotion, climbing, and head positionbias. To directly inhibit the excitatory STN neurons, we deliveredlentiviruses carrying eNpHR under the CaMKIIα promoter to the right STNof the hemiparkinsonian rats. CaMKIIα::eNpHR-EYFP expression wasspecific to excitatory neurons (as shown by CaMKIIα and glutamateexpression; FIG. 4B, right; FIG. 11A), robust (95.73%±1.96 s.e.minfection rate assessed in n=220 CaMKIIα positive cells), and restrictedto the STN (FIG. 4B, left and middle). To validate the resultingphysiological effects of optical control, a hybrid opticalstimulation/electrical recording device (optrode) was employed inisoflurane-anesthetized animals to confirm that eNpHR was functional invivo, potently inhibiting (>80%) spiking of recorded neurons in the STN(FIG. 4C; FIG. S13A, B; FIG. 14A). This cell type-targeted inhibitionwas temporally precise and reversible, and extended across all frequencybands of neuronal firing (FIG. 4C, FIG. 16A).

For behavioral rotation assays in the hemiparkinsonian rats, theSTN-targeted fiberoptic was coupled to a 561nm laser diode to driveeNpHR. Electrical DBS was highly effective at reducing pathologicalrotational behavior, but despite precise targeting and robustphysiological efficacy of eNpHR inhibition, the hemiparkinsonian animalsdid not show even minimal changes in rotational behavior with directtrue optical inhibition of the local excitatory STN neurons (FIG. 4D).In addition, there was no effect on path length and head position biasin response to light during these experiments (see supplementarymethods). While muscimol and lidocaine administration to the region ofthe STN in monkeys and rodents can relieve Parkinsonian symptoms (30),the data in FIG. 4 show that the more specific intervention ofselectively decreasing activity in excitatory local neurons of the STNappeared not sufficient by itself to affect motor symptoms.

Another possibility is that DBS could be driving secretion of glialmodulators which would have the capability to modulate local STNcircuitry; this would be consistent with recent findings indicating thata glial-derived factor (adenosine) accumulates during DBS and plays arole in DBS-mediated attenuation of thalamic tremor. Indeed, the STNexpresses receptors for glia-derived modulators which can inhibitpostsynaptic currents in the STN. ChR2 presents an interestingpossibility for recruitment of glia; when opened by light, in additionto Na⁺ and K^(±) ions, ChR2 can also pass trace Ca²⁺ currents thattrigger Ca²⁺ waves in and activate ChR2-expressing astroglia. Weemployed a GFAP promoter to target ChR2 to local astroglia, validatedwith GFAP and S100β staining (FIG. 5A, FIG. 11B). Optrode recordingsrevealed that blue light stimulation of STN following transduction withGFAP::ChR2 reversibly inhibited neuronal firing in the STN (FIG. 5B,FIG. 12A), with variable delay on the timescale of seconds. However,recruiting astroglial cells by this mechanism was not sufficient tocause even trace responses in motor pathology in parkinsonian rodents(FIG. 5C, FIG. 12B). Path length and head position bias were also notaffected by light during these experiments. While these data do notexclude the importance of local STN inhibition as a contributing factorin DBS response, as not all STN neurons may be affected in the same wayby indirect glial modulation, the direct activation of local glial cellsappeared not sufficient to treat parkinsonian symptoms, pointing toother circuit mechanisms.

Network oscillations at particular frequencies could play importantroles in both PD pathology and treatment. For example, PD ischaracterized by pathological levels of beta oscillations in the basalganglia, and synchronizing STN at gamma frequencies may ameliorate PDsymptoms while beta frequencies may worsen symptoms. Because simpleinhibition of excitatory cell bodies in the STN did not affectbehavioral pathology, and since high-frequency stimulation (HFS: 90-130Hz) is used for electrical DBS, we used ChR2 to drive high-frequencyoscillations in this range within the STN. We injected CaMKIIα::ChR2into the STN (FIG. 6A) and used pulsed illumination with a 473nm laserdiode to activate excitatory neurons in the STN (FIG. 6B, FIG. 14B)during behavioral testing in parkinsonian rodents (FIG. 6C, FIG. 12C).Despite robust effects on high-frequency power of neuronal spike rate inSTN of anesthetized animals (FIG. 16B), HFS delivered locally to the STNarea failed to affect PD behavioral symptoms (path length and headposition bias were unchanged by light—see supplementary methods).Animals tested in parallel with beta frequency pulses also showed nobehavioral response, indicating that (while not excluding a contributoryrole) directly generated oscillations within the STN excitatory neuronsare not sufficient to account for therapeutic effects.

We have previously measured in cortical and hypothalamic tissue thepropagation of blue light in the setting of laser diode-fiberopticillumination; we observed that substantial tissue volumes (comparable tothat of the STN) could reliably be recruited at a light power densitysufficient to drive physiologically significant microbial opsincurrents. It was important to repeat and extend these measurements tothe PD setting. First, we confirmed that the propagation measurements ofblue light (473 nm) in brain tissue represent a lower bound on thevolume of tissue recruited, due to reduced scattering of lower-energyphotons delivered from the 561 nm laser diode; therefore sufficientlight power is present to activate opsins within 1.5 mm of the fiber foreither wavelength of light (FIG. 7A). We next extended these findingswith a functional assay for tissue recruitment under conditionsmimicking our behavioral experiments (FIG. 7B,C). After an in vivooptical stimulation paradigm targeted to the CaMKIIα::ChR2 expressingSTN in freely moving rats, we performed immunohistochemistry for c-fos,a biochemical marker of neuronal activation. We observed robust c-fosactivation in STN (FIG. 7B) over a widespread volume (FIG. 7C); indeed,as expected from our light scattering measurements and tissue geometry,we found that at least 0.7 mm³ of STN is recruited by light stimulation,closely matching the actual volume of the STN (FIG. 7C). Therefore,light penetration was not limiting since the entire STN is recruited bythe optical modulation paradigms of FIGS. 4-6.

Therapeutic effects could arise from driving axonal projections thatenter the STN, as DBS electrodes will potently modulate not just localcells and their efferents, but also afferent fibers. Optogeneticsdiscriminates these two possibilities, as the lentiviruses transducesomata without transducing afferent axons. To assess the possibilitythat PD motor behavioral responses are modulated by targeting afferentprojections to the STN, we used Thy1::ChR2 transgenic mice in which ChR2is expressed in projection neurons, and we verified that in Thy1::ChR2line 18, ChR2-YFP is excluded from cell bodies in the STN but isabundant in afferent fibers (FIG. 8A).

We conducted optrode recordings in anesthetized 6-OHDA mice (FIG. 10B)to assess local effects on STN physiology of driving afferent axonsselectively, and found frequency-dependent effects (FIG. 8B). First, weobserved that HFS of afferent fibers to the STN potently reduced STNspiking across all frequency bands; this effect did not completely shutdown local circuitry, as low-amplitude high-frequency oscillationspersisted during stimulation (FIG. 8B; FIG. 13C, D; FIG. 14C). Next, wefound that LFS of afferent fibers increased beta-frequency firing in theSTN without affecting endogenous bursting (FIG. 8B, FIG. 14D). We nextassessed the impact of these specific interventions on PD behavior in6-OHDA mice, and for the first time among the optogenetic interventions,we observed marked effects. Driving STN afferent fibers with HFSrobustly and reversibly ameliorated PD symptoms, measured by rotationalbehavior and head position bias (FIG. 8C). The HFS effects were notsubtle; indeed, in nearly every case these severely parkinsonian animalswere restored to behavior indistinguishable from normal, and in everycase the therapeutic effect immediately and fully reversed, with returnof ipsilateral rotations upon discontinuation of the light pulseparadigm. Notably, treated animals could freely switch directions ofmovement and head position from left to right and vice versa. Instriking contrast with optical HFS, optical LFS (20 Hz) of the sameafferent fibers worsened PD symptoms by driving increased ipsilateralrotational behavior (FIG. 8C), demonstrating that behavioral effectsseen do not result from simply driving unilateral activity. Therefore,in contrast to direct STN cellular interventions, driving STN afferentfibers with HFS and LFS differentially modulated PD symptoms in a mannercorresponding to frequencies of stimulation linked clinically toameliorated or exacerbated PD symptoms.

A diverse array of fibers from widespread brain areas converge on theSTN, perhaps underlying the utility of the STN as a focal DBS target.Many of these afferents likely contribute together to the therapeuticeffects, and it is unlikely that a single source of fibers completelyaccounts for the behavioral effects seen. However, we explored theseafferents in greater detail to determine the general class of fibersthat may be contributory.

Thy1::ChR2 animals display ChR2 expression chiefly in excitatoryprojection neurons. Indeed, the inhibitory markers GAD67 and GABA werenot detectable in Thy1::ChR2 fibers within STN (FIG. 9A, left),effectively ruling out contributions from the GABAergic pallidalprojections (LGP/GPe). We also found no localization of majorneuromodulatory markers (dopamine and acetylcholine) within the STNThy1::ChR2 fibers (FIG. 11C), ruling out dopaminergic SNr as a relevantfiber origin as well. We next explored possible sources of excitatoryfibers, and found no expression of ChR2-YFP in the cell bodies of theexcitatory parafascicular or pedunculopontine nuclei, potentialcontributors of excitatory fibers to the STN. Within neocortex of thesemice, however, ChR2-YFP is expressed strongly in excitatory neurons thatproject to STN. Since pathologically strong connectivity between STN andprimary motor cortex M1 has been suggested to underlie PD circuitdysfunction, we therefore explored M1 as a possible contributor.

We verified in Thy1::ChR2 M1 the presence of strong and selective ChR2expression largely restricted to layer V neurons and correspondingapical dendrites but not in cells within other layers (FIG. 9A, right).To probe the functional connectivity between these layer V projectionneurons and STN in the PD animals, we conducted a separated-optrodeexperiment in anesthetized animals in which the fiberoptic and recordingelectrodes were placed in two different brain regions in Thy1::ChR2animals (FIG. 9B). By driving M1 layer V projection neurons andsimultaneously recording in both M1 and STN, we found that precise M1stimulation of this kind potently influenced neural activity in the STN(FIG. 9C, FIG. 16C, D), and that M1 Layer V neurons could beantidromically recruited by optical stimulation in the STN (FIG. 15).While as noted above, many local afferents in the STN region, includingfrom the ZI, are likely to underlie the complex therapeutic effects ofDBS, functional influences between M1 layer V and STN could be asignificant contributor. Indeed, we found that selective M1 layer V

HFS optical stimulation sufficed to ameliorate PD symptoms in a similarmanner to STN stimulation in an array of measures ranging fromrotational behavior (FIG. 9D) to head position bias and locomotion (FIG.9E, F). As with STN stimulation, pathological rotations and headposition bias were reduced by optical HFS to M1; in contrast, while notaugmenting the pathology, optical 20 Hz (LFS) stimulation to M1 had notherapeutic effect (FIG. 9D, E, F), and even at the highest lightintensities achievable without epileptogenesis, M1 LFS did not drive ormodify rotational behavior, unlike M2 LFS cortical stimulation that canelicit contralateral rotations. Finally, increased functional mobilitywith M1 HFS but not LFS was confirmed with quantification of increaseddistance and speed of locomotion in PD Thy1::ChR2 mice; in the absenceof amphetamine, M1 HFS allowed the otherwise bradykinetic animals tomove freely without eliciting rotational behavior (FIG. 9F).

FIG. 4 shows direct optical inhibition of local STN neurons. (A) Cannulaplacement, virus injection, and fiber depth were guided by recordings ofthe STN, which is surrounded by the silent zona incerta (ZI) andinternal capsule (IC). (B) Confocal images of STN neurons expressingCaMKIIα::eNpHR-EYFP and labeled for excitatory neuron-specific CaMKIIα(right). (C) Continuous 561nm illumination of the STN expressingCaMKIIα::eNpHR-EYFP in anesthetized 6-OHDA rats reduced STN activity;representative optrode trace and amplitude spectrum shown. Mean spikingfrequency was reduced from 29±3 Hz to 5±1 Hz (mean±s.e.m., p<0.001,Student's t-test, n=8 traces from different STN coordinates in 2animals). (D) Amphetamine-induced rotations were not affected bystimulation of the STN in these animals (p>0.05, n=4 rats, t-test withp=0). The red arrow indicates direction of pathologic effects, while thegreen arrow indicates direction of therapeutic effects. The electricalcontrol implanted with a stimulation electrode showed therapeuticeffects with HFS (120-130 Hz, 60 μs pulse width, 130-200 μA, p<0.05,t-test with p=0). Percent change of −100% indicates that the rodent isfully corrected. Data in all figures are mean±s.e.m. ns p>0.05, *p<0.05,**p<0.01, ***p<0.001.

FIG. 5 shows targeting astroglia within the STN. (A) Confocal imagesshow STN astrocytes expressing GFAP::ChR2-mCherry, costained with GFAP(right). (B) 473 nm illumination of the STN expressingGFAP::ChR2-mCherry in anesthetized 6-OHDA rats. Optrode recordingrevealed that continuous illumination inhibited STN activity with 404±39ms delay to onset and 770±82 ms delay to offset (n=5 traces fromdifferent STN coordinates in 2 animals), while 50% duty cycle alsoinhibited spiking, with delay to onset of 520±40 ms and delay to offsetof 880±29 ms (n=3 traces from different STN coordinates in 2 animals)with p<0.001. (C) Amphetamine-induced rotations were not affected by 50%duty cycle illumination in these animals (right, p>0.05, n=7 rats,t-test with p=0).

FIG. 6 shows optical depolarization of STN neurons at differentfrequencies. (A) Confocal images of STN neurons expressingCaMKIIα::ChR2-mCherry and labeled for the excitatory neuron specificCaMKIIα marker. (B) Optical HFS (120 Hz, 5 ms pulse width) of the STNexpressing CaMKIIα::ChR2-mCherry in 6-OHDA rats recorded with theoptrode connected to a 473nm laser diode (representative trace andamplitude spectrum shown). Frequency of spiking increased from 41±2 Hzto 85±2 Hz (HFS vs. pre, n=5 traces: p<0.001, t-test, post, n=3 traces;traces were sampled from different STN coordinates in 1 animal). (C)Amphetamine-induced rotations were not affected by high (left, 130 Hz, 5ms, n=5 rats) or low (middle, 20 Hz, 5 ms, n=2 rats) frequency opticalstimulation.

FIG. 7 shows quantification of the tissue volume recruited by opticalintervention. (A) Intensity values for 473 nm (blue) and 561nm (yellow)light are shown for a 400 μm fiber as a function of depth in braintissue. The dashed line at 1 mW/mm² (30 mW light source) indicates theminimum intensity required to activate channelrhodopsins andhalorhodopsins. (B) Confocal images of STN neurons expressingCaMKIIα::ChR2-mCherry and labeled for the immediate early gene productc-fos show robust neuronal activation produced by light stimulation invivo. Arrowheads indicate c-fos positive cells. Freely moving ratsexpressing ChR2 in STN (same animals as in FIG. 6), were stimulated with473 nm light (20 Hz, 5 ms pulse width). (C) The STN volume that showedstrong c-fos activation was estimated to be at least 0.7 mm³ (dashedlines indicate STN boundaries); robust c-fos activation was observedmedial-lateral (1.155 mm), anterior-posterior (0.800 mm), anddorsal-ventral (0.770 mm) on subthalamic slices imaged by confocalmicroscopy with DAPI counterstain.

FIG. 8 shows selective optical control of afferent fibers in the STN.(A) Confocal images of Thy1::ChR2-EYFP expression in the STN and DAPIstaining for nuclei shows selective expression in fibers and not cellbodies (right). (B) Optical HFS (130 Hz, 5 ms pulse width) of the STNregion in an anesthetized Thy1::ChR2-EYFP 6-OHDA mouse with 473nm lightinhibited STN large-amplitude spikes (sample trace, top left), whileinducing smaller-amplitude high-frequency oscillations (FIGS. 13C, D;14C). Optical LFS (20 Hz, 5 ms pulse width) produced reliable spiking at20 Hz (bottom left). While HFS prevented bursting (top right, p<0.001,n=3), LFS had no significant effect on burst frequency by 2 samplet-test (p>0.05, n=3 traces) nor on spikes/burst (bottom right, p>0.05,n=3 traces). (C) Optical HFS to STN in these animals (left, 100-130 Hz,5 ms, n=5 mice) produced robust therapeutic effects, reducingipsilateral rotations and allowing animals to freely switch directions.In contrast, optical LFS (second left, 20 Hz, 5 ms, n=5 mice)exacerbated pathologic effects, causing increased ipsilateral rotations.Both effects were reversible (Post). Changes were significant by t-testwith μ=0 for both HFS (p<0.001, n=5 mice) and LFS (p<0.05, n=5 mice)compared to baseline (light off). (F) Contralateral head position biasalso showed robust correction with HFS by 2 sample t-test (HFS vs. lightoff: p<0.05; n=2 mice), but not with LFS (LFS vs. light off: p>0.05, n=2mice).

FIG. 9 shows selective optical stimulation of layer V neurons inanterior primary motor cortex. (A) GAD67 and GABA staining showed nocolocalization with Thy1::ChR2-EYFP in STN (left). Apical dendrites oflayer V neurons can be seen rising to the pial surface (22, 23) (right).(B) Schematic for optical stimulation of M1 with simultaneous recordingin STN of Thy1::ChR2 mice. (C) Optical stimulation (473nm) of M1 andsimultaneous recording in STN of anesthetized Thy1::ChR2 mice. OpticalHFS (130 Hz, 5 ms pulse width) of M1 modulated activity in both M1 andSTN. Optical LFS (20 Hz, 5 ms) of M1 produced 20 Hz tonic firing in bothM1 and STN. (D) Optical HFS (130 Hz, 5 ms pulse width) reducedamphetamine-induced ipsilateral rotations in 6-OHDA Thy1::ChR2 mice(p<0.01, n=5 mice) in contrast to optical LFS (20 Hz, 5 ms pulse width,p>0.05, n=4 mice); t-test with p=0. (E) Contralateral head position biaswas corrected in HFS (HFS vs. light off: p<0.001, n=4 mice), while LFShad little effect (LFS vs. light off: p>0.05, n=3 mice); 2-samplet-test. (F) HFS but not LFS to M1 significantly increased path length(HFS, p<0.01, n=2 mice) and climbing (p<0.05, n=3 mice); 2-samplet-test. Sample paths before, during, and after HFS are shown (100seconds each, path lengths noted in cm).

According to a specific implementation, the following steps followed inobtaining the results discussed herein. To verify the phenotype of cellsand measure c-fos activity, rodents were anaesthetized with 65 mg/kgsodium pentobarbital and transcardially perfused with ice-cold 4%paraformaldehyde (PFA) in PBS (pH 7.4). Brains were fixed overnight in4% PFA and then equilibrated in 30% sucrose in PBS. 40 μm-thick coronalsections were cut on a freezing microtome and stored in cryoprotectantat 4° C. until processed for immunohistochemistry. Free-floatingsections were washed in PBS and then incubated for 30 min in 0.3% TritonX-100 (Tx100) and 3% normal donkey serum (NDS). Slices were incubatedovernight with primary antibody in 0.01% Tx100 and 3% NDS (rabbitanti-cfos 1:500, rabbit anti-GFAP 1:500, mouse anti-MAP2 1:500, mouseanti-GAD67 1:500, rabbit anti-GABA 1:200, mouse anti-vGlut1 1:500, mouseanti-vGlut2 1:500, mouse anti-CaMKIIα 1:200, mouse anti-S100β 1:250,rabbit anti-glutamate 1:200, chicken anti-tyrosine hydroxylase 1:500,and goat anti-choline acetyltransferase 1:200). Sections were thenwashed and incubated with secondary antibodies (1:1000) conjugated toFITC, Cy3 or Cy5 for 3 hrs at room temperature. Following a 20 minincubation with DAPI (1:50,000) sections were washed and mounted onmicroscope slides with PVA-DABCO.

Confocal fluorescence images were acquired on a scanning lasermicroscope using a 2033 /0.70NA or a 40×/1.25NA oil immersion objective.To determine the volume of c-fos activation, serial stack imagescovering a depth of 20 pm through multiple medial-lateral,anterior-posterior and dorsal-ventral subthalamic sections were acquiredusing equivalent settings. The image analysis software calculated thenumber of c-fos positive cells per field by thresholding c-fosimmunoreactivity above background levels and using the DAPI staining todelineate nuclei. To determine the rate of viral transduction wedetermined the percentage of CaMKIIα-immunoreactive neurons per 40Xfield that were also eNpHR-YFP positive in multiple serial stack imagesof subthalamic sections. Large field images of entire slices werecollected on a Leica MZ16FA stereomicroscope.

Lentiviral vectors carrying the genes used were constructed usingcloning techniques. The CaMKIIα::eNpHR construct was produced by PCRamplification of the eNpHR-EYFP construct previously published andcloned in-frame into the AgeI and EcoRI restriction sites of alentivirus carrying the CaMKIIα promoter. The CaMKIIα::ChR2 constructwas produced by PCR amplification of the ChR2-mCherry construct and wasalso cloned in-frame into the AgeI and EcoRI restriction sites of alentivirus carrying the CaMKIIα promoter. The GFAP::ChR2 construct wasproduced by replacing the CaMKIIα promoter with the GFAP promoter in theCaMKIIα::ChR2-mCherry construct using the AgeI and PacI restrictionenzyme sites. High titer lentivirus (>10⁹ pfu/mL) was then produced viacalcium phosphate co-transfection of 293FT cells with the lentiviralvector, pCMVAR8.74 and pMD2.G (S2). 24 h post-transfection, 293 FT cellswere switched to serum-free medium containing 5 mM sodium butyrate; thesupernatant was collected 16 h later and concentrated byultracentrifugation at 50,000×g with 20% sucrose cushion. The resultingviral pellet was resuspended in phosphate buffered saline at 1/1000th ofthe original volume.

To ensure that there would be no significant expression leak innon-targeted cell types, we employed a Cre-inducible AAV vector with adouble-floxed inverted open reading frame (ORF), wherein the ChR2-EYFPsequence is present in the antisense orientation. Upon transduction,Cre-expressing cells invert the ChR2-EYFP ORF in a stable, irreversiblefashion and thereby activate sustained ChR2-EYFP expression undercontrol of the strong and constitutively active elongation factor 1α(EF-1α) promoter (Feng Zhang, unpublished results). To constructCre-activated recombinant AAV vectors, the DNA cassette carrying twopairs of incompatible lox sites (loxP and 1ox2722) was synthesized andthe ChR2-EYFP transgene was inserted between the loxP and 1ox2722 sitesin the reverse orientation. The resulting double-floxed reverseChR2-EYFP cassette was cloned into a modified version of the pAAV2-MCSvector carrying the EF-1α promoter and the Woodchuck hepatitis virusposttranscriptional regulatory element (WPRE) to enhance expression. Therecombinant AAV vectors were serotyped with AAV5 coat proteins andpackaged by the viral vector core at the University of North Carolina.The final viral concentration was 2×10¹² genome copies (gc)/mL.

Adult rats (female Fisher, 200-300 g) and mice (male and female, C57BL/6background, 15-30 g) were the subjects of these experiments. Animalhusbandry and all aspects of experimental manipulation of our animalswere in strict accord with guidelines from the National Institute ofHealth and have been approved by members of the Stanford InstitutionalAnimal Care and Use Committee. All surgeries were performed underaseptic conditions. Rodents were anaesthetized using 1.5% isoflurane(for surgeries longer than 1 hr) or i.p. injection (90 mg/kg ketamineand 5mg/kg xylazine for rats; 80 mg/kg and 15-20 mg/kg, respectively,for mice). The top of the animal's head was shaved, cleaned with 70%ethanol and betadine and then placed in a stereotactic apparatus.Ophthalmic ointment was applied to prevent eye drying. A midline scalpincision was made and then small craniotomies were performed using adrill mounted on the stereotactic apparatus for the 6-OHDA injection inthe medial forebrain bundle (rat: −2 AP, 2 ML, −7.5 DV; mouse: −1.2 AP,1.2 ML, −4.75 DV) and virus injection in the STN (rat: −3.6 mm AP, 2.5mm ML; mouse: −1.9 mm AP, 1.7 mm ML).

For rodents that were injected with lentivirus in the STN, in vivoextracellular recording was used to accurately determine the location ofthe STN along the dorsal-ventral axis. The depth was around −7 mm inrats and −4 mm in mice. The concentrated lentivirus (described above)was delivered to the STN using a 10 μl syringe and a thin 34 gauge metalneedle; the injection volume and flow rate (3 sites within the STN alongthe dorsal-ventral axis; each injection was 0.6 μl at 0.1 μl/min) wascontrolled with an injection pump. After the final injection, the needlewas left in place for 10 additional minutes and then slowly withdrawn.

6-OHDA was then used to lesion the substantia nigra and producehemi-Parkinsonian rodents. Desipramine (20 mg/kg for rats; 10 mg/kg formice; noradrenergic reuptake inhibitor to prevent damage tonoradrenergic terminals) was administered, followed ˜30 minutes later by6-OHDA (8 μg/4 μl for rats; 6 μg/2 μl for mice) with 0.1% ascorbic acid(to prevent degradation of 6-OHDA) into the right medial forebrainbundle (rat: −2 AP , +2 ML, and -7.5 DV; mouse: −1.2 AP, +1.2 ML, and−4.75 DV). The perfusion for the 6-OHDA injection (rat: 4 mouse 2 μl)was at the rate of 1.2 μl/min for 4 min, and the needle was left in situfor an additional 5 minutes.

A fiber guide (rat: C312G, mouse: C313G) was beveled to form a sharpedge (to more easily penetrate brain tissue and reduce tissue movement),and then inserted through the craniotomy to a depth of approximately 400μm above the STN or the anterior primary motor cortex (mouse: 2 AP, 2ML, 0.5 DV). One layer of adhesive cement followed by cranioplasticcement was used to secure the fiber guide system to the skull. After 20min, the scalp was sealed back using tissue adhesive. The animal waskept on a heating pad until it recovered from anesthesia. Buprenorphine(0.03 mg/kg) was given subcutaneously following the surgical procedureto minimize discomfort. A dummy cannula (rat: C312G, mouse: C313G) wasinserted to keep the fiber guide patent. For electrical DBS controlrodents, a stimulation electrode (MS303/3-B) was implanted in the STN.The procedure above was followed for OHDA injection, in vivoextracellular recording was then used to determine the depth of the STN,and the stimulation electrode was inserted to that depth and securedusing one layer of adhesive cement followed by cranioplastic cement.Tissue adhesive was used to reseal the scalp, the animal was kept on aheating pad until recovery from anesthesia and buprenorphine was givento minimize discomfort. A dust cap (303DC/1) was then used to cover theelectrode contacts.

Simultaneous optical stimulation and electrical recording in a singleregion in living rodents was done as described previously using anoptrode composed of an extracellular tungsten electrode (1 MΩ, ˜125 μm)tightly attached to an optical fiber (˜200 μm) with the tip of theelectrode deeper (˜0.4 mm) than the tip of the fiber, to ensureillumination of the recorded neurons. For stimulation and recording intwo distinct regions, small craniotomies were created above both targetregions, and a fiber or optrode was placed above one region through onecraniotomy and a plain electrode or optrode was placed in the otherregion through a separate craniotomy (see FIG. 9B for diagram).Stimulation in the anterior motor cortex was achieved by placing theoptical fiber just above the brain surface, activating layer 5 of thecortex; for STN stimulation, the fiber was 300 μm above the STN. The STNwas identified using its highly stereotyped firing pattern and the factthat it is surrounded dorso-ventrally by silent regions. The opticalfiber was coupled to a 473 nm or 561 nm laser diode (30 mW fiber output)from CrystaLaser. Single unit recordings were done in rats anesthetizedwith 1.5% isoflurane and mice anesthetized with intraperitonealinjections of ketamine (80 mg/kg)/xylazine (15-20 mg/kg) cocktail.pClamp 10 and a Digidata 1322A board were used to both collect data andgenerate light pulses through the fiber. The recorded signal was bandpass filtered at 300Hz low/5 kHz high (1800 Microelectrode ACAmplifier). For precise placement of the fiber/electrode pair,stereotactic instrumentation was used.

For behavior, multimode optical fibers (NA 0.37; rat: 400 μm core,BFL37-400; mouse: 300 μm core, BFL37-300) were precisely cut to theoptimal length for maximizing the volume of the STN receiving light.About one week before behavior, an extracellular recording electrode wasused to determine the depth of the dorsal border of the STN from the tipof the cannula guide and fibers were cut to be 200-300 μm shorter. Foranterior motor cortex stimulation, the fiber was placed above layer 5(less than a millimeter deep). To ensure stability of the fiber duringtesting in moving animals, an internal cannula adapter was glued to thestripped optical fiber. To insert the fiber, rodents were briefly placedunder isoflurane and the fiber was inserted while the animal wasrecovering from anesthesia. The internal cannula adapter snapped ontothe cannula guide and the bottom half of the plastic portion of a dummycannula was also used to ensure the adapter remained connected to thetop of the cannula guide.

For optical stimulation, the fiber was connected to a 473 nm or 561 nmlaser diode (20 mW fiber output) through an FC/PC adapter. Laser outputwas controlled using a function generator (33220A) to vary thefrequency, duty cycle, and intensity. For Thy1::ChR2 animals, theaverage minimum intensity used to produce therapeutic behavior was 10mW. A custom aluminum rotating optical commutator was used to releasetorsion in the fiber caused by the animal's rotation.

Motor behavior was assessed using amphetamine-induced rotations, headposition bias, climbing, and track length Animals were accepted forexperimental investigation only if amphetamine reliably inducedrotations in the ipsilateral direction confirming a 6-OHDA lesion of thesubstantia nigra. Before and after each stimulation trial, a trial ofequal length with the light off was used as a control. Each of thesetrials was about 3 minutes long making the entire off-on-off sequence 9minutes long. For amphetamine-induced behavior, amphetamine (rat: 2mg/kg; mouse: 2.6 mg/kg) was injected 30 minutes before behavioralmeasurements; the fiber was inserted into the cannula and the rodentplaced in an opaque, non-reflective cylinder (rat: diameter 25 cm,height 61 cm; mouse: diameter 20 cm, height 46 cm) 10 minutes before thebehavioral experiments. Rotations ipsilateral to the 6-OHDA lesions(clockwise turns) were counted, and contralateral rotations weresubtracted. The percentage change calculation considered the change inrotational bias relative to the period without stimulation. Headposition bias was determined by counting the number of head tilts (>10°deviation left or right of midline) over time. Each time the rodent roseup and touched either paw to the wall of the cylinder was counted as aninstance of climbing. Track length was measured with Viewer. After thecompletion of behavior experiments, cannula placement was confirmed byslicing.

For experiments where optical stimulation did not produce a change inthe rodent behavior, we also collected path length and head positionbias data while the rodents were under amphetamine. Continuous 561nmillumination of the STN expressing CaMKIIα::eNpHR-EYFP in 6-OHDA ratsdid not affect path length (cm/min; light on vs. light off:757.05±163.11 vs. 785.74±157.56, p=0.90, n=4 rats; mean±s.e.m; 2-samplet-test) or head position bias (% of time to the right; light on vs.light off: 99.92±0.08 vs. 99.75±0.25, p=0.56, n=4 rats; mean±s.e.m;2-sample t-test). Optical HFS (120 Hz, 5 ms pulse width) or LFS (20 Hz,5 ms pulse width) of the STN expressing CaMKIIα::ChR2-mCherry in 6-OHDArats did not affect path length (cm/min; HFS vs. light off:803.82±129.04 vs. 851.95±166.20, p=0.83, n=5 rats; LFS vs. light off:847.15±141.95 vs. 779.11±104.01, p=0.74, n=2 rats; mean±s.e.m; 2-samplet-test) or head position bias (% of time to the right; HFS vs. lightoff: 93.97±3.78 vs. 94.20 ±2.96, p=0.96, n=5 rats; LFS vs. light off:98.50±1.50 vs. 98.50±0.50, p=1.00, n=2 rats; mean±s.e.m; 2-samplet-test). 473nm illumination of the STN expressing GFAP::ChR2-mCherry in6-OHDA rats also did not affect path length (cm/min; light on vs. lightoff: 1042.52±113.73 vs. 1025.47±113.63, p=0.92, n=4 rats; mean±s.e.m;2-sample t-test) or head position bias (% of time to the right; light onvs. light off: 98.16 ±0.98 vs. 98.98±0.65, p=0.52, n=4 rats; mean±s.e.m;2-sample t-test).

Light transmission measurements were conducted with blocks of braintissue prepared from two 300 g Fisher rats and immediately tested.Blocks of tissue 2 mm in thickness were cut in 0-4 ° C. sucrose solutionusing a vibratome. The tissue was then placed in a Petri dish containingthe same sucrose solution over the photodetector of a power meter. Thetip of a 200 pm diameter optical fiber coupled to a blue or yellow diodelaser (473 nm or 561 nm, 30 mW fiber output) was mounted on amicromanipulator. First, the power was measured through the solution.Then, the tip of the fiber was moved down into the tissue in 100 μmincrements and the power was measured. When the fiber reached the Petridish, the power measured was compared to the initial measurement throughthe solution to confirm the total power output through the fiber. Thepercent transmission fraction was then calculated as the ratio betweenthe power measured through tissue and the power measured throughsolution. The power intensity was then calculated by considering thelight intensity spread due to the conical shape of the 30 mW lightoutput from a 400 μm fiber based on the fiber's numerical aperture of0.37. The fiber output was assumed to be uniform across the diameter ofthe cone. Measurements were made through grey matter in three blocks ofbrain tissue for each wavelength with one block each movinganterior-posterior in the thalamus and in the cortex and dorsal-ventralthrough the thalamus.

Threshold search in Clampfit was used for automated detection of spikesin the multi-unit recording, which was then validated by visualinspection; the spike waveforms displayed by Clampfit were observed tocheck the quality of spike detection. For traces with multiple spikepopulations, thresholds were set to capture all the spikes; duringbursting, it is likely that multiple neurons were recorded fromsimultaneously. Bursts were identified in Clampfit; any two consecutivespikes occurring in an interval less than 300 ms were counted asbelonging to the same burst and only bursts of at least 3 spikes wereincluded. To quantify the neural activity at different frequencies,spectra for in vivo extracellular recording traces were generated usinga wavelet transform after converting the traces into binary spiketrains. The trace was then converted into a histogram with a binwidth of0.5 ms for each of the duration-matched pre-stimulation, stimulation,and post-stimulation epochs. The start and end times for each of thesegments, as well as the number of spikes, are listed below.

TABLE 1 The three segments of each power spectra were time matched; thistable shows the segments of each trace (the start and end time inseconds), as well as the number of spikes detected during each period.Time intervals were chosen to reflect stationary states before, during,and after stimulation for each trace, to account for temporal delays inonset or offset of physiological effects. Pre-Stimulation Light OnPost-Stimulation Start End Spikes Start End Spikes Start End Spikes FIG.4 32.5 72.5 413 102.5 142.5 84 175 215 435 (CaMKIIα::eNpHR) FIG. 7 5.2810.4 238 15.38 20.5 477 22.48 27.6 235 (CaMKIIα::ChR2, HFS) FIG. 8 010.62 90 14.98 25.6 0 29.38 40 94 (Thy1::ChR2, HFS) FIG. 8 (Thy1::ChR2,0 10.62 139 14.98 25.6 383 29.38 40 132 LFS) FIG. 9 (Thy1, HFS 0.8 4.855 15.46 19.46 28 26 30 30 M1, M1 recd) FIG. 9 (Thy1, HFS 0.94 5.4 19 1519.46 37 25.54 30 16 M1, STN recd) FIG. 9 (Thy1, LFS 0 5.5 131 18.5 24313 30.5 36 64 M1, M1 recd) FIG. 9 (Thy1, LFS 0 5.5 50 18.5 24 115 30.536 39 M1, STN recd) FIG. 13 (eNpHR, 32.5 72.5 263 102.5 142.5 84 175 215248 small unit) FIG. 13 (eNpHR, 32.5 72.5 114 102.5 142.5 0 175 215 145large unit)

The spike histograms were then convolved with a wavelet to measure theamplitude of the spectra at frequencies below 150 Hz over time. Theaverage amplitude over time for each frequency was then plotted. Thewavelet used is reproduced below.

g(f,t)=e ^(−t) ² ^(/2σ) ² e ^(−2πift)

σ=4/(3f).

For determining the change in activity of multiple frequency bands,amplitude spectra for multiple duration-matched baseline and stimulationsweeps were calculated as described above. Mean amplitude within eachfrequency band was determined and the ratio of this value(stimulation/baseline) was calculated. Spike latencies of the M1response to optical stimulation of the STN were determined by measuringthe delay between the first peaks in simultaneous optrode recordings ofM1 and STN of a Thy1::ChR2-EYFP 6-OHDA mouse. 20 Hz, 5 ms pulse width of473 nm light was used to activate the STN.

FIG. 10 shows substantia nigra lesion and cannula track. Loss of nigraldopaminergic cells following 6-OHDA administration in rat (A) and mouse(B): coronal slices (rat: AP −5.8; mouse AP −3) show decreased tyrosinehydroxylase levels (red) unilaterally in the substantia nigra parscompacta; SNc is outlined by white brackets. Insets below show higherresolution images of the lesioned (left) and unlesioned (right) sides ofthe substantia nigra. (C) Cannula track is visible in a coronal sliceshowing correct placement of the cannula above the STN area.

FIG. 11 shows an additional histological characterization. (A) STN cellsexpressing CaMKIIα::eNpHR-EYFP (green) label for the excitatory neuronspecific glutamate marker (red). (B) STN cells expressingGFAP::ChR2-mCherry (red) costain with the astroglia-specific markerS100β (green). In both (A) and (B) yellow indicates colocalization ofthe two markers. (C) Representative confocal images of TH stain fordopamine (top) and CHAT stain for acetylcholine (bottom) showed nocolocalization with Thy1::ChR2-EYFP expression in the STN.

FIG. 12 shows additional behavioral results. (A) Continuous 473nmillumination of the STN expressing GFAP::ChR2-mCherry in an anesthetized6-OHDA mouse completely inhibited STN activity. (B) and (C): Extensionof mouse results. (B) Amphetamine-induced rotations were not affected by50% duty cycle illumination of the GFAP::ChR2 expressing STN in 6-OHDAmice (n=1 mouse and 2 sessions). (C) Amphetamine-induced rotations werenot affected by high (130 Hz, 5 ms pulse width, n=1 mouse and 2sessions) or low (20 Hz, 5 ms, n=1 mouse and 1 session) frequencyoptical stimulation in the CaMKIIα::ChR2 expressing STN in 6-OHDA mice.(D) and (C): Modulation of inhibitory neurons during behavior. Althoughmainly excitatory, STN has about 7-10% percent cells that stain forinhibitory neuronal markers, such as GAD65/67 and parvalbumin (AllenBrain Atlas). To obtain specific expression in either GAD67 orparvalbumin neurons we injected GAD67-Cre and parvalbumin-Cre micerespectively (gift of Sylvia Arber) with a Cre-inducibleadeno-associated virus (AAV) vector carrying ChR2-EYFP (Methods).Cre-dependent opsin expression was observed in the STN region, butbehavior was unchanged with optical stimulation. (D) Amphetamine-inducedrotations were not affected by high (130 Hz, 5 ms, n=2 mice and 4sessions) or low (20 Hz, 5 ms, n=1 mouse and 2 sessions) frequencyoptical stimulation in 6-OHDA GAD67-Cre mice. (E) Amphetamine-inducedrotations were not affected by high (130 Hz, 5 ms, n=2 mice and 2sessions) or low (20 Hz, 5 ms, n=2 mice and 2 sessions) frequencyoptical stimulation in 6-OHDA parvalbumin-Cre mice.

FIG. 13 shows additional electrophysiological results. Isolation oflarge amplitude (A) and small amplitude (B) units from the trace in FIG.4C and corresponding power spectra. Red lines represent averagewaveforms for all superimposed spikes that occurred during 70s ofbaseline activity (n=205 spikes for large amplitude unit and n=428spikes for small amplitude unit). Both small and large amplitude unitsshowed decreased activity during light that returned to normal baselinelevels after stimulation. (C) Response of STN to optical stimulation ofSTN in a Thy1::ChR2-EYFP 6-OHDA mouse at 90Hz. The STN is initiallyexcited but activity is reduced in the emergent stationary statemeasured by loss of the large amplitude spikes evident during thebaseline; nevertheless, significant low amplitude activity persiststhroughout the stimulation. (D) High-temporal resolution trace of theSTN response to optical stimulation of STN in a Thy1::ChR2-EYFP 6-OHDAmouse at 130Hz (see FIG. 5B for full trace). Again, the STN initiallyresponds with a spike followed by low amplitude activity throughoutstimulation. Changes in amplitude of the local circuit responses canreflect either altered recruited cell number or altered excitability ofrecruited cellular elements. While optrode recordings cannot report onthe precise cell types involved in generating activity, by eliminatingthe electrical stimulation artifact these recordings provide a windowinto the amplitude and timing properties of local circuit electricalresponses arising from local excitatory or inhibitory cell types andfibers in the STN region that could not be achieved with electricalstimulation.

FIG. 14 shows high-temporal resolution optrode traces. (A) Single unitactivity in CaMKIIα::eNpHR-EYFP expressing STN with continuous 561 nmlight illumination in an anesthetized 6-OHDA rat (corresponding to tracein FIG. 4C). (B) Neuronal activity in CaMKIIα::ChR2-mCherry expressingSTN with high frequency optical stimulation (120 Hz, 5 ms pulse width,473 nm) in an anesthetized 6-OHDA rat (corresponding to trace in FIG.3B). (C) and (D) Activity in the STN region in an anesthetizedThy1::ChR2-EYFP 6-OHDA mouse in response to high (HFS, 130 Hz, 5 ms) andlow (LFS, 20 Hz, 5 ms) frequency optical stimulation using 473 nm light.Note the low amplitude of activity in the HFS trace (corresponding totrace in FIG. 5B).

FIG. 15 shows latency of M1 response to optical stimulation of STN. (A)Response of M1 Layer 5 (L5) to optical stimulation of STN in theThy1::ChR2-EYFP 6-OHDA mouse at 20Hz, 5ms pulse width. (B) Whilestimulating STN with light, simultaneous recordings of light-inducedactivity in the STN (top trace) and M1/L5 (bottom trace) revealed shortlatency differences between the first peaks consistent with antidromicspiking. (C) Individual latency differences between the first peak inSTN and M1/L5 for 16 stimulation bouts revealed minimal jitter(S.D.=0.032 ms) consistent with antidromic spiking in the well-knownM1-STN projection.

FIG. 16 shows changes in frequency characteristics of neuronal activityproduced by optical stimulation. (A) Activity in all frequency bands wasreduced by continuous 561 nm illumination of the STN expressingCaMKIIα::eNpHR-EYFP in anesthetized 6-OHDA rats (n=5 sweeps). Frequencybands are defined as: delta 1-3 Hz; theta 4-8 Hz; alpha 9-12 Hz; beta13-30 Hz; gamma 31-80 Hz; high frequency (HF) 81-130 Hz. (B) Optical HFS(120 Hz, 5 ms pulse width) of the STN expressing CaMKIIα::ChR2-mCherryin 6-OHDA rats reduced activity for frequencies between 4 and 80 Hz,while increasing activity in the HF band (n=3). (C) Activity change inM1 (left, n=4) and STN (right, n=4) produced by optical HFS (130 Hz, 5ms) stimulation of M1 in 6-OHDA Thy1::ChR2 mice. Delta activity in bothM1 and STN was reduced. (D) Activity change in M1 (left, n=4) and STN(right, n=4) produced by optical LFS (20 Hz, 5 ms) stimulation of M1 in6-OHDA Thy1::ChR2 mice. Beta, gamma, and HF activity in both M1 and STNwas increased. (E) Optical LFS (20 Hz, 5 ms) of the STN in 6-OHDAThy1::ChR2 mice increased activity in the beta, gamma, and HF bands(n=3). (F) Spike counts for duration-matched baseline and opticalstimulation segments for each experiment type. Optical stimulation ofthe STN expressing CaMKIIα::GFAP-mCherry and optical HFS in 6-OHDAThy1::ChR2 mice abolished spiking activity, reducing activity across allfrequencies to zero (not shown). Error bars are s.e.m.; t-test withμ=100 used for statistics, *p<0.05.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Based on the above discussion and illustrations, those skilled in theart will readily recognize that various modifications and changes may bemade to the present invention without strictly following the exemplaryembodiments and applications illustrated and described herein. Forinstance, such changes may include additional modifications toVChR1-based sequences. Such modifications and changes do not depart fromthe true spirit and scope of the present invention, which is set forthin the following claims.

1-22. (canceled)
 23. A method for treating Parkinson's Disease in anindividual, the method comprising: a) implanting into the individual adevice comprising: i) a recombinant expression vector comprising anucleotide sequence encoding a Volvox carteri light-activated ionchannel (VChR1) protein to provide localized depolarization of afferentaxons of the subthalamic nucleus (STN); and ii) a light-deliverymechanism, wherein the VChR1 protein is produced in the afferent axons;and b) activating the VChR1 protein with the light-delivery mechanism,thereby depolarizing the afferent axons and treating the disease. 24-29.(canceled)
 30. The method of claim 23, wherein the VChR1 proteinexhibits excitation in a range of from about 535 nm to about 589 nm. 31.The method of claim 23, wherein the VChR1 protein comprises the aminoacid sequence set forth in SEQ ID NO:3.
 32. The method of claim 23,wherein the nucleotide sequence operably linked to an alpha-CaMKIIpromoter.
 33. The method of claim 23, wherein the nucleotide sequencethat is codon optimized for expression in a mammalian cell.
 34. Themethod of claim 23, wherein the light delivery mechanism is alight-emitting diode.
 35. The method of claim 23, wherein the lightdelivery mechanism is a fiber optic.
 36. The method of claim 23, whereinthe light delivery mechanism is a laser.
 37. The method of claim 23,wherein the expression vector is a viral vector.
 38. The method of claim37, wherein the viral vector is a lentivirus vector, a retroviralvector, or an adenoassociated virus vector.
 39. A treatment methodcomprising: a) implanting into an individual a device comprising: i) arecombinant expression vector comprising a nucleotide sequence encodinga Volvox carteri light-activated ion channel (VChR1) protein; and ii) alight-delivery mechanism, wherein the VChR1 protein is produced in acell in the individual; and b) activating the VChR1 protein with thelight-delivery mechanism, thereby depolarizing the cell and treating theindividual.
 40. The method of claim 39, wherein the VChR1 proteinexhibits excitation in a range of from about 535 nm to about 589 nm. 41.The method of claim 39, wherein the VChR1 protein comprises the aminoacid sequence set forth in SEQ ID NO:3.
 42. The method of claim 39,wherein the nucleotide sequence operably linked to an alpha-CaMKIIpromoter.
 43. The method of claim 39, wherein the nucleotide sequencethat is codon optimized for expression in a mammalian cell.
 44. Themethod of claim 39, wherein the light delivery mechanism is alight-emitting diode.
 45. The method of claim 39, wherein the lightdelivery mechanism is a fiber optic.
 46. The method of claim 39, whereinthe light delivery mechanism is a laser.
 47. The method of claim 39,wherein the expression vector is a viral vector.
 48. The method of claim47, wherein the viral vector is a lentivirus vector, a retroviralvector, or an adenoassociated virus vector.
 49. The method of claim 39,wherein the cell is a neuron or a muscle cell.
 50. The method of claim39, wherein individual has a neurological disease, and whereinactivating the VChR1 protein with the light-delivery mechanism treatsthe neurological disease.